Compositions and Methods for Paraffin Liquefaction and Enhanced Oil Recovery in Oil Wells and Associated Equipment

ABSTRACT

The subject invention provides compositions comprising solvents and surfactants, as well as their use in improving oil and/or gas production. In some embodiments, the compositions comprise chemical or synthetic solvents and/or surfactants. In other embodiments, the compositions comprise biological components, such as microorganisms and/or their growth by-products. The subject invention can be used to dissolve, disperse and/or emulsify paraffin precipitates and/or deposits; prevent and/or inhibit paraffin deposition; remove rust deposits and prevent corrosion associated therewith; inhibit bacterial growth and/or biofilm formation; and to enhance oil recovery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/052,181, filed Oct. 31, 2020; which is a National Stage Applicationof International Application No. PCT/US2019/029870, filed Apr. 30, 2020;which claims priority to U.S. Provisional Patent Application Ser. No.62/664,613, filed Apr. 30, 2018; Ser. No. 62/682,462, filed Jun. 8,2018; Ser. No. 62/691,098, filed Jun. 28, 2018; Ser. No. 62/719,734,filed Aug. 20, 2018; Ser. No. 62/743,815, filed Oct. 10, 2018; and Ser.No. 62/805,539, filed Feb. 14, 2019; each of which are incorporated byreference herein in their entireties.

BACKGROUND OF THE INVENTION

The safe and efficient production of hydrocarbon compositions depends onthe proper functioning of hydrocarbon-producing facilities. One of themost common issues leading to structural failure and productioninefficiency is the formation of deposits in and around the wellbore,tubing, flow lines, storage tanks, separators, and other components ofoil and gas production infrastructure, as well as in the pores of thereservoir rock.

These problematic deposits can be formed by, for example,high-molecular-weight constituents of petroleum fluids, most notably,paraffins and asphaltenes, as well as bacterial deposits, often in theform of biofilms.

Paraffin deposits, in particular, can range from soft accumulations oflighter-molecular-weight paraffins to hard and/or brittle accumulationsas the molecular weight of the paraffin increases. Paraffin depositionis primarily a result of a loss in solubility of certain components inthe crude oil, which can be caused by a decrease in temperature orpressure in an oil well or formation.

Temperature decrease can be caused by, for example, expansion of gasthrough perforations and from the lifting of fluids to the earthsurface; radiation of heat from tubing into the surrounding formation;intrusion of water into or around the wellbore; vaporization-inducedloss of lighter constituents in the crude oil; and/or recovery andtransport of oil during winter months or in climates that are coldyear-round. Additionally, in offshore operations, decrease intemperature can occur when crude oil enters subsea pipelines, which canhave temperatures as low as 4° C.

Along with temperature decrease, pressure decrease can impact thecomposition of crude oil, which causes loss of volatiles and inducesprecipitation of paraffins. For this reason, mature oil wells commonlyexperience paraffin deposition issues. As the production time of a wellcontinues and lighter components of crude oil are depleted, leavingbehind heavier fractions, reservoir pressure and flow rate of oildecrease. Furthermore, films and chemicals build up with time in thepores of the shale, reducing hydrocarbon movement into the wellbore.This can lead to changes in temperature and/or pressure gradients andthus greater paraffin accumulation.

The “pour point” and the “cloud point” are two physical properties ofliquid fuels that can also contribute to the precipitation of dissolvedsolids such as paraffins in crude fluids, as well as the ability ofcrude fluids to flow through pipelines and tubulars. Cloud point (or,Wax Appearance Temperature) is the temperature at which a cloud of waxcrystals first appear in a liquid fuel. Above the cloud point, dissolvedsolids present in the crude remain soluble, but below the cloud point,they start to precipitate and create a cloudy appearance. This is anindicator of how well the fuel will perform under cold weatherconditions, because, not only can wax clog equipment, but it can alsodeposit onto pipelines and other equipment surfaces.

Pour point is the lowest temperature at which the flow or movement ofoil, i.e., the ability of the oil to be pumped, is still possible. Belowthis temperature, the crude stops flowing and starts to crystallizeand/or freeze. Highly paraffinic crude oils have higher pour pointvalues.

Paraffin that remains entrained in crude oil does not typically causeissues in production. It is when the paraffin particles precipitate andbegin to accumulate as solid or semi-solid deposits that the mostsignificant problems related to paraffin occur. The presence of water onthe formation pore walls or on tubing inner surfaces, as well as thequality and smoothness of pipes can inhibit deposition; however rustypipes with rough surfaces will encourage deposition. Thus, upkeep ofequipment surfaces is important for prevention of paraffin depositions.Furthermore, minimizing the cooling of the crude oil as it is brought tothe surface, using, for example specially designed pumping wells andtubing can aide in prevention of paraffin depositions.

Paraffin inhibitor chemicals, which are a class of compounds thattypically consist of crystal modifiers that prevent the deposition ofparaffin onto surfaces, can also be used. These surface-active materialsinhibit the adhesion of paraffin to sites on, e.g., the tubing walls;however, the efficacy of any given inhibitor chemical depends upon thespecific composition of the crude oil within the well, which can behighly variable depending on, for example, the geographical location.Other methods of inhibition, involving plastic coatings on tubulars, andelectrical heaters, can be extremely costly, and thus are limited.

Once even a thin layer of paraffin deposit is formed on a surface, therate of further accumulation drastically increases. Thus, systematictreatment or removal of deposits is crucial to maintaining properlyfunctioning oil producing facilities. As the thickness of depositsincreases over time, the result is a gradual decrease in production. Intubing and casing structures, the deposits begin to reduce the innerdiameter of piping and restrict the free flow of oil and gas. As thisoccurs, the interior roughness of the structures also increases, whichraises the pump pressure required to move the petroleum product. If leftuntreated, deposits can ultimately lead to complete blockage.Furthermore, depending upon the location of the precipitation,maintenance and/or emergency repairs can become extremely expensive.

Current methods of deposit removal fall within four main categories:mechanical, chemical, microbial, and thermal removal. Mechanical removaltypically involves the use of scrapers or cutters to physically removedeposits. For example, in tanks where precipitation has occurred, thesides of the tank must be cut out and force, e.g., a sledgehammer, isthen used to remove the deposits. For pipelines, complete replacement ofpipes is often required if deposits become too thick for manual ormechanical removal.

Chemical removal involves the use of solvents or surfactants that cansolubilize deposits or interfere with their crystallization andformation. Examples of widely-used solvents include benzene, toluene andxylene. With microbial methods, certain strains of bacteria can be usedto degrade deposits themselves, or can produce natural biochemicals thatdo so.

Along with many of these methods, however, thorough removal of depositsoften requires the addition of some type of thermal treatment. Thermalremoval, with steam, hot water or hot oil, for example, is useful formelting or dissolving deposits, and as noted, for supplementing othermethods of removal. This requires high energy inputs, however, and theuse of hot steam can be dangerous for workers at the site ofapplication. Furthermore, the liquefaction of paraffin is often onlytemporary, meaning the paraffin will almost immediately re-solidify dueto the properties of the oil and/or the environment. This is aparticular problem with highly-paraffinic crudes, e.g., those recoveredfrom Utah formations, and could also become a problem in Permianformations when temperatures drop during the winter.

Biofilms can also build up in various structures and processingmechanisms, including shale formation facing, wells, pipes, and tanks.“Biofilm” comprises layers of biomass made up of a compact grouping ofmicroorganisms surrounded by an extracellular matrix of polymericsubstances. Biofilms adhere to surfaces of many man-made mechanisms,such as tubes and pipes, and can significantly impair their properfunctioning. Additionally, many of the biofilms present in, or on, oilrigs contain sulfate-reducing bacteria that generate potent chemicalbyproducts, e.g., hydrogen sulfide. Hydrogen sulfide gas is harmful fordrill workers who might breathe it. Furthermore, hydrogen sulfide cancause corrosion of various mechanisms within an oil producing structure(known as “microbial induced corrosion” or “MIC”) and can cause thesouring of oil during storage or transport. Sour oil contains a highsulfur content, which increases costs for producers and consumers due tothe increase of time and resources required for processing the oil.

Accumulation of organic deposits in and on oil processing equipment andin the pores of oil-bearing formations can have a compounding effect.Unless these organic compounds are removed, operators can be faced withlowering yields, improper function of pumps, corroded or blocked tubingand pipes, and potential for total loss of production. Furthermore,cost, safety in processing, large-scale sustainability, and damage toformations must be accounted for when developing methods for removingthese deposits to ensure long-term efficiency of hydrocarbon production.

Because of the importance of safe and efficient oil and gas productionand the difficulties caused by paraffin deposits in production andtransport of oil and gas, there is a continuing need for improvedmethods of removing these deposits from oil-bearing formations andassociated production equipment.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides compositions and methods for improvingoil well performance by removing paraffin deposits from oil- and/ornatural gas-bearing formations, and/or the wells and productionequipment associated therewith, as well as for enhancing oil recovery.

In certain embodiments, materials and methods are provided for improvingoil and/or gas production by liquefying or dissolving solid paraffindeposits and dispersing and/or emulsifying precipitated paraffin backinto crude oil. Advantageously, in one embodiment, the paraffin remainsdispersed in the oil after treatment and does not re-precipitate.

In preferred embodiments, the subject invention provides a compositionfor improving oil and/or gas production, the composition comprising oneor more solvents and one or more surfactants.

In certain embodiments, the composition comprises one or more solvents,one or more surfactants, one or more yeast fermentation products, one ormore chelating agent(s), and, optionally, one or more ammonium saltsand/or co-surfactants.

In one embodiment, the solvent(s) and/or the surfactant(s) can beproduced by non-biological means (e.g., chemical isolation, purificationand/or synthesis). In another embodiment, the solvents and/orsurfactants can be derived from natural or biological sources, such as,for example, the living cells of microorganisms, plants, fungi and/oranimals.

In one embodiment, the composition can further comprise one or moreyeast fermentation products. In one embodiment, a yeast fermentationproduct comprises a yeast strain, such as, for example, Wickerhamomycesanomalus, Starmerella bombicola, or Meyerozyma guilliermondii, and/orby-products produced during cultivation of the yeast. In certainembodiments, the yeast cells are thermally inactivated before beingadded to the composition.

In certain embodiments, use of yeast fermentation products according tothe subject invention can be superior to, for example, purifiedmicrobial metabolites alone, due to, for example, the advantageousproperties of the yeast cell walls. These properties include highconcentrations of mannoprotein as a part of yeast cell wall's outersurface (mannoprotein is a highly effective bioemulsifier) and thepresence of biopolymer beta-glucan (also an effective emulsifier) inyeast cell walls. Additionally, the yeast fermentation product furthercan comprise biosurfactants capable of reducing both surface andinterfacial tension, enzymes capable of solubilizing heavy hydrocarbonand/or paraffinic compounds, and other metabolites (e.g., lactic acid,ethyl acetate, ethanol, etc.), in the culture.

In some embodiments, certain fungi, other than yeasts, have cell wallscontaining the same advantageous properties. Accordingly, fermentationproducts comprising non-yeast fungi can also be used according to thesubject invention.

In one embodiment, a first yeast fermentation product, designated as“Star 3+,” can be obtained via cultivation of a yeast, e.g.,Wickerhamomyces anomalus, using a modified form of solid statefermentation. The culture can be grown on a substrate with ample surfacearea onto which the yeasts can attach and propagate, such as, forexample, corn flour, rice, soybeans, chickpeas, pasta, oatmeal or beans.The entire fermentation medium with yeast cells growing throughout, andany growth by-products thereof (e.g., enzymes, solvents, and/orbiosurfactants) can be harvested after, for example, 3-5 days ofcultivation at 25-30° C. The culture can be washed out and used inliquid form, or blended with the solid substrate, milled and/ormicronized, and optionally, dried. This comprises the Star 3+ product.The product can be diluted in oil, water and/or brine fluids, forexample, 5 to 1,000 times, prior to being added to the composition.

In an alternative embodiment, the first yeast fermentation product(e.g., Star 3+) is obtained using submerged fermentation, wherein thefirst yeast fermentation product comprises liquid broth comprisingwater, cells and any yeast growth by-products.

In one embodiment, the composition according to the subject inventioncomprises one or more solvents. Preferably, the one or more solvents arenot produced by the yeasts of the first yeast fermentation product,meaning they are present in addition to any solvents that may happen tobe present in the first yeast fermentation product.

In one embodiment, the one or more solvents are non-polar aromaticsolvents. In one embodiment, the solvents can include one or more of,for example, terpenes, terpenoids, acetates, ionic or semi-ionicliquids, alcohols, kerosene, gasoline, diesel, benzene, toluene, and/orxylene.

In one embodiment, the one or more solvents can includenaturally-derived acetates, such as, for example, isoamyl acetate and/orprimary amyl acetate.

In one embodiment, the one or more solvents can include terpenes and/orterpenoids, such as, for example, turpentine, dipentene and/orD-limonene.

In one embodiment, the one or more solvents can include alcohols, suchas hexanol and/or isopropyl alcohol.

In one embodiment, the one or more solvents can include an ionic orsemi-ionic liquid, for example, a semi-ionic liquid comprising a mixtureof glycerol and Epsom salt (MgSO₄.7H₂O).

In one embodiment, any combination of these solvents is utilized withone or more surfactants. In one embodiment, the composition iscustomized based on the type of paraffin that is present in a well. Forexample, paraffin deposits can vary from soft accumulations to hard,brittle, solidified deposits. Thus, in some embodiments, theconcentration of solvent in the composition can be increased (e.g., upto about 10% of the composition) to boost the dispersal capabilitieswhen harder paraffins are present.

In one embodiment, the composition comprises one or more surfactants,which, along with paraffin removal and/or dispersal, can provideadditional enhanced oil recovery. The surfactant(s) can be ofnon-biological origin and/or they can be biosurfactants, meaningsurfactants produced by a living cell. Non-biological surfactants can beselected from, for example, anionic, cationic, zwitterionic and/ornonionic classes of surfactants.

In a specific embodiment, the one or more surfactants arebiosurfactants. Preferably, the one or more biosurfactants are notproduced by the yeasts of the first yeast fermentation product, meaningthey are included in the composition in addition to any biosurfactantsthat may be present in the first yeast fermentation product.

In certain embodiments, the biosurfactants can be added to thecomposition in purified form and/or in crude form. In certainembodiments, the biosurfactant can be added to the composition in theform of a microbial culture, e.g., a second yeast fermentation product,containing liquid fermentation broth and cells resulting from submergedcultivation of a biosurfactant-producing microbe, e.g., Wickerhamomycesanomalus, Starmerella bombicola or Meyerozyma guilliermondii.

In some embodiments, a blend of biosurfactants is used. Biosurfactantsuseful according to the subject invention include, for example,low-molecular-weight glycolipids, cellobiose lipids, lipopeptides, fattyacid esters, fatty acid ethers, flavolipids, phospholipids, andhigh-molecular-weight polymers/biopolymers such as lipoproteins,lipopolysaccharide-protein complexes, and/orpolysaccharide-protein-fatty acid complexes.

In one embodiment, the biosurfactants can comprise one or moreglycolipids such as, for example, rhamnolipids (RLP),rhamnose-d-phospholipids, trehalose lipids, trehalose dimycolates,trehalose monomycolates, mannosylerythritol lipids (MEL), cellobioselipids, ustilagic acids and/or sophorolipids (SLP) (including lactonicforms and/or acidic forms). In one embodiment, the biosurfactants cancomprise one or more lipopeptides, such as, for example, surfactin,iturin, fengycin, arthrofactin, viscosin, amphisin, syringomycin, and/orlichenysin. In one embodiment, the biosurfactants can comprise one ormore fatty acid esters and/or one or more fatty acid ethers. In oneembodiment, the biosurfactants can comprise one or more other types ofbiosurfactants, such as, for example, cardiolipin, emulsan, lipomanan,alasan, and/or liposan.

In one embodiment, the surfactants can comprise one or more microbialcompounds having physical properties and/or behaviors similar to thoseof biosurfactants, but which are not commonly known as biosurfactants.These compounds can be fatty acid esters and/or fatty acid ethers. Incertain embodiments, the fatty acid compounds can comprise carbon chainswith 6 to 22 carbon atoms. In certain embodiments, the fatty acid(s) ofthe fatty acid compounds is unsaturated.

In one embodiment, the composition further comprises one or morechelating agents, for example, EDTA, citric acid, citrate, sodiumacetate, or a mixture thereof. In specific embodiments, the chelatingagent is sodium citrate.

The subject composition can further comprise carriers (e.g., water, oiland/or brine fluids) as well as other compounds that are useful forparaffin removal and/or enhanced oil recovery, such as, for example,ammonium salts, co-surfactants, and/or enzymes (e.g., extracellularenzymes derived from Aspergillus spp.), These additional compounds canbe added at concentrations ranging from, for example, about 0.001% to50%, about 1% to 25%, or about 10%, by weight or volume.

Advantageously, the compositions of the subject invention are shelfstable for at least one week or longer, and can be transported, storedand then applied selectively to an oil well at any point, for example,after a decline in production is observed.

In certain embodiments, the subject invention provides methods ofimproving oil and/or gas production, wherein a composition according tothe subject invention is applied to a subterranean formation, an oiland/or gas well, a wellbore, and/or equipment associated therewith. Insome embodiments, the methods improve the efficiency of oil and/or gasrecovery by, e.g., decreasing the amount of resources and energyrequired to recover oil and/or gas from a formation, and in general,increasing the amount of oil and/or gas recovered over a certain periodof time.

In one embodiment, the methods improve oil and/or natural gas(hereinafter, “gas”) production through the removal and dispersal ofparaffin deposits and/or precipitates that have accumulated in asubterranean formation, in an oil and/or gas well, in a wellbore and/orin production equipment associated with any of these. For example, themethods are useful for removing paraffin deposits from the rock pores ofsubterranean formations, from wells, wellbores, and from equipment, suchas, for example, tubing, pipes, drills and tanks associated with allaspects of oil and/or gas production.

Additionally, the methods provide for recovery of economically valuableparaffin hydrocarbons by dispersing and/or emulsifying the dislodgedparaffin back into crude oil fluids. Advantageously, applying thesubject composition also helps inhibit paraffin deposition, and helpsprevent re-deposition of dispersed paraffins while pumping andtransporting.

The composition can be customized for a particular well. Thus, in oneembodiment, the method comprises testing the well and/or associatedequipment, analyzing the paraffin composition present therein anddetermining the ideal formulation for the composition prior totreatment. Advantageously, the subject methods can be useful for removaland dispersal of a broad spectrum of paraffin types, including shortchain paraffins and long chain paraffins.

Even further, in addition to ultimately increasing the amounts of crudeoil recovered from a well due to the clearing and/or dispersing ofparaffin deposits, the methods also enhance oil recovery through, forexample, the amphiphilic properties of surfactants, includingbiosurfactants.

The subject methods can also be useful for a multitude of other benefitsrelated to oil and gas recovery, including, for example: inhibition ofparaffin crystallization and prevention of paraffin deposition;reduction in viscosity of paraffinic crude oil; reduction in pour pointof paraffinic crude oil (e.g., to about −25° F./−32° C.); removal and/ordissolution of scale; release of rust from oilfield casings and relatedequipment; protection against under-deposit rust-related corrosion ofequipment; inhibition of bacterial growth and disruption of biofilmformation on equipment; protection against microbial induced corrosion(MIC); alteration of the wettability of the near-wellbore surface towater-wet; and remediation of formation skin damage.

In one embodiment, the subject methods can be used alongside and/or toenhance or supplement other methods of paraffin removal/dispersal and/orenhanced oil recovery, e.g., other microbial, mechanical, thermal and/orchemical treatments.

The method can be used to replace dangerous high heat steaming or oilingmethods commonly used for paraffin removal. When, however, thermal,steaming and/or hot oil methods are used, the present method can be usedalongside (before, during or after) the thermal, steaming and/or hot oilto prevent recrystallization of the liquefied paraffins that aredispersed in the oil.

In one embodiment, the subject methods comprise a chemical treatment forremoving paraffin deposits present, for example, at or near a wellbore.The chemical treatment method can comprise applying a composition thatcomprises one or more chemical solvents and one or more non-biologicalsurfactants to the wellbore. Advantageously, the combination of solventswith surfactants, when compared to using, for example, solvents alone,can also provide enhanced oil recovery in addition to effectivelydispersing paraffin back into crude oil fluids. Additionally, thischemical treatment does not require large volumes of treatment mixture,as is required for treating deep into a subterranean formation. Thesurfactants can comprise, for example, from 1% to 50% of the volume ofthe chemical treatment, from 2% to 20%, or from 5% to 10%.

In one embodiment, the subject methods can be utilized alongside and/orin combination with enzyme treatments for removal of hydrocarbondeposits and/or enhanced oil recovery, e.g., extracellular enzymesderived from Aspergillus spp.

In certain embodiments, the subject invention can be used for improving,enhancing, and/or maintaining oil recovery from, and operation of,subterranean formations, oil and/or gas wells, boreholes, tubes, pipes,drills, tanks and other structures and equipment involved in oil and/orgas production, transportation, storage and refining. The subjectinvention can be used in, for example, vertical, horizontal and/orfracking wells, mature wells, stripper (marginal) wells, flowlines, toclean near wellbore zones and to clean storage tanks.

In one embodiment, application of a composition of the subject inventioncan be performed during drilling operations (e.g., while drilling, whiletripping-in or tripping-out of the hole, while circulating mud, whilecasing, while placing a production liner, and/or while cementing, etc.),and/or as a production treatment (e.g., after oil and/or gas recovery isunderway). In some embodiments, the methods are implemented once therate of oil production from the well has begun to decline and/or at somepoint thereafter.

Advantageously, in certain embodiments, the subject compositions andmethods can free stuck or floating rods, allowing inoperable wells toresume operation. Furthermore, in one embodiment, the subject treatmentscan open up channels and pores/pore throats that are clogged withparaffin deposits and the adhesive/cohesive matrices that form whenscale, polymers, sand, and other materials become lodged in theparaffin, thus allowing for improved oil production. Even further, thesubject treatments require lower frequencies of application whencompared to other conventional paraffin treatment methods.

Advantageously, the subject invention can be utilized in recovery andtransport of oil in locations where lower temperatures might causeparaffin deposition, such as, for example, in offshore wells, in thearctic or Antarctic, and in climates that experience cold wintertemperatures.

Furthermore, the subject invention can be utilized in oil wells withhigh formation water salinity levels. For example, the compositions canbe useful in geologic regions where formation water salinity is up to250,000 ppm (total dissolved solids), up to 300,000 ppm, or even up to400,000 ppm or more.

Even further, the compositions can be useful in treating mature wellsand wells that have undergone hot oiling, as well as for removal ofshort- and long-chain paraffin deposits, including those that areparticularly difficult to remove due to, for example, the heaviness,thickness and/or the hardness of the deposit.

In one embodiment, the subject compositions and methods can be usedwithout releasing large quantities of inorganic compounds into theenvironment. Additionally, the compositions and methods can utilizecomponents, such as biosurfactants, that are biodegradable andtoxicologically safe. Thus, while the subject invention can utilizenon-biological or synthetic chemical components, the present inventioncan also be formulated as an environmentally-friendly treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows pour point test tube treatment of paraffin with acomposition according to one embodiment of the subject invention. Thebottom of the test tube contains a sold paraffin portion (denoted by thedashed arrow), which became solid at −3° C. and did not flow upontilting the test tube. D-limonene and canola oil separated from thesolution while the mixture was being chilled and remained liquid(denoted by the solid arrow).

FIGS. 2A-2B show dispersal of a hard paraffin after 2 hours and 4 hourswith two formulations of a composition according to embodiments of thesubject invention. The formulation used in (B) comprised double theamount of solvent that the formulation used in (A) comprises.

FIGS. 3A-3B show dispersal of a hard paraffin after 1 hour using acomposition according to one embodiment of the subject invention (A), ascompared to other treatments (B) (water, pentane, xylene, kerosene,condensate).

FIGS. 4A-4B show dispersal of a hard paraffin after 2 hours using acomposition according to one embodiment of the subject invention (A), ascompared to other treatments (B) (water, pentane, xylene, kerosene,condensate).

FIGS. 5A-5B show dispersal of a hard paraffin after 1 hour using acomposition according to one embodiment of the subject invention (A), ascompared to other treatments (B) (water, pentane, xylene, kerosene,condensate).

FIGS. 6A-6B show a segment of corroded casing from an oil well in theAppalachian region before treatment (A) and after treatment (B) with acomposition according to one embodiment of the subject invention. Asshown, the amount of rust on the segment was reduced after 16 hours ofsoaking in the subject composition.

FIGS. 7A-7B show (A) Gram-positive and (B) Gram-negative bacterialinhibition in CFU/ml over time after treatment with a compositionaccording to one embodiment of the subject invention. The darkest,highest line indicates the normal growth of the respective bacteria(control).

FIGS. 8A-8C show a comparison of biofilm formation inhibition withdirect treatment with 1% (A), 5% (B) and 10% v/v (C) dilutions of thesubject composition versus control, untreated biofilm. Direct treatmentinvolved adding a composition according to one embodiment of the subjectinvention directly to the surface of the bacteria.

FIGS. 9A-9C show a comparison of biofilm formation inhibition withindirect treatment with 1% (A), 5% (B) and 10% v/v (C) dilutions of acomposition according to one embodiment of the subject invention versuscontrol, untreated biofilm. Indirect treatment involved removing asection of agar from a plate where bacterial culture was spread anddried, injecting the composition into the voided space in the agar, andallowing the composition to soak into the agar.

FIGS. 10A-10B show the effect of a composition according to oneembodiment of the subject invention on disrupting established biofilm(B) versus untreated control (A). Within 16 hours of treatment, theinitial signs of biofilm degradation could be observed.

DETAILED DESCRIPTION

The subject invention provides compositions and methods for improvingoil well performance by removing paraffin deposits from oil- and/ornatural gas-bearing formations, and/or the wells and productionequipment associated therewith, as well as for enhancing oil recovery.

In certain embodiments, materials and methods are provided for improvingoil and/or gas production by liquefying or dissolving solid paraffindeposits and dispersing and/or emulsifying precipitated paraffin backinto crude oil. Advantageously, in one embodiment, the paraffin remainsdispersed in the oil after treatment and does not re-precipitate.

The subject methods can also be useful for a multitude of other benefitsrelated to oil and gas recovery, including, for example: inhibition ofparaffin deposition; release of rust from oilfield casings and relatedequipment; protection against under-deposit rust-related corrosion ofequipment; inhibition of bacterial growth and disruption of biofilmformation on equipment; protection against microbial induced corrosion(MIC); reduction in viscosity of paraffinic crude oil; reduction in pourpoint of paraffinic crude oil (e.g., to about −25° F./−32° C.); removaland/or dissolution of scale; alteration of the wettability of thenear-wellbore surface to water-wet; and remediation of formation skindamage.

Selected Definitions

As used herein, “contaminant” refers to any substance that causesanother substance or object to become fouled or impure. Contaminants canbe living or non-living and can be inorganic or organic substancesand/or deposits. Furthermore, contaminants can include, but are notlimited to, hydrocarbons, such as petroleum, tar sands or asphaltenes;fats, oils and greases (FOG), such as cooking grease and lard; lipids;waxes, such as paraffin; resins; biofilms; or any other substancesreferred to as, for example, dirt, dust, scale (including calciumcarbonate, calcium chloride, barium carbonate, barium chloride, and ironsulfide), sludge, crud, slag, grime, scum, plaque, buildup, or residue.

In preferred embodiments of the subject invention, the contaminant isparaffin. According to the subject invention, paraffins include anywax-like organic hydrocarbon precipitate belonging to the alkane groupand having a general formula of C_(n)H_(2n+2). They can include normal,branched or cyclic alkanes. Further, they can include shorter chain(e.g., 20 carbons) to longer chain (e.g., 40 carbons or more) paraffins.In some instances, deposited paraffin also contains mixtures of gums,resins, asphaltic material, polymers, crude oil, scale, sand, silt,water and other formation substances. They vary in consistency from amushy liquid to a firm hard wax, depending upon, for example, the amountand type of oil present, the temperature, and the age of the deposit.

As used herein, “removal” as used in the context of contaminants orfouling means elimination or reduction of contaminants from a surface, aspace or a piece of equipment. Removal can include purifying, defouling,decontaminating, clearing or unclogging, and can be achieved by anymeans, including but not limited to, liquefying, dissolving, melting,dispersing, emulsifying, scraping, degrading, blasting, soaking, orcleaving the contaminant. Furthermore, removal can be total or partial.

As used herein, “prevention” means avoiding, delaying, forestalling,inhibiting or minimizing the onset or progression of an occurrence orsituation. Prevention can include, but does not require, absolute orcomplete prevention, meaning the occurrence or situation may stilldevelop, but at a later time than it would without preventativemeasures. Prevention can also include reducing the severity and/orextensiveness of an occurrence or situation, and/or inhibiting theprogression in severity and/or extensiveness. In certain embodiments,the subject invention can be useful for preventing the deposition and/orre-deposition of paraffin onto a surface.

As used herein, reference to a “microbe-based composition” means acomposition that comprises components that were produced as the resultof the growth of microorganisms or other cell cultures. Thus, themicrobe-based composition may comprise the microbes themselves and/orproducts of microbial growth. The microbes may be in a vegetative state,in spore form, in mycelial form, in any other form of propagule, or amixture of these. The microbes may be planktonic or in a biofilm form,or a mixture of both. The products of growth may be, for example,metabolites, cell membrane components, expressed proteins, and/or othercellular components. The microbes may be intact or lysed; active orinactive. In some embodiments, the microbes are present, with medium inwhich they were grown, in the microbe-based composition. The microbesmay be removed from the composition, or they may be present at, forexample, a concentration of at least 1×10⁴, 1×10¹, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ or more propagules permilliliter of the composition. As used herein, a propagule is anyportion of a microorganism from which a new and/or mature organism candevelop, including but not limited to, cells, spores (e.g., reproductivespores, endospore and exospores), mycelia, cysts, conidia, buds andseeds.

The subject invention further provides “microbe-based products,” whichare products that are to be applied in practice to achieve a desiredresult. The microbe-based product can be simply the microbe-basedcomposition harvested from the microbe cultivation process.Alternatively, the microbe-based product may comprise furtheringredients that have been added, or it may have ingredients removedtherefrom. Additional ingredients can include, for example, stabilizers,buffers, appropriate carriers, such as water, salt solutions, or anyother appropriate carrier, added nutrients to support further microbialgrowth, non-nutrient growth enhancers, such as plant hormones, and/oragents that facilitate tracking of the microbes and/or the compositionin the environment to which it is applied. The microbe-based product mayalso comprise mixtures of microbe-based compositions. The microbe-basedproduct may also comprise one or more components of a microbe-basedcomposition that have been processed in some way such as, but notlimited to, filtering, centrifugation, lysing, drying, purification andthe like.

As used herein, an “isolated” or “purified” nucleic acid molecule,polynucleotide, polypeptide, protein or organic compound, such as asmall molecule, is substantially free of other compounds, such ascellular material, with which it is associated in nature. A purified orisolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid(DNA)) is free of the genes or sequences that flank it in itsnaturally-occurring state. A purified or isolated polypeptide is free ofother molecules, or the amino acids that flank it, in itsnaturally-occurring state.

As used herein, reference to an isolated microbe strain means that thestrain is removed from the environment in which it exists in nature.Thus, the isolated strain may exist as, for example, a biologically pureculture, or as spores (or other forms of the strain) in association witha carrier.

In certain embodiments, purified compounds are at least 60% by weightthe compound of interest. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight the compound of interest. For example, a purified compound is onethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w)of the desired compound by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 20 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 and 20, as well as all intervening decimal values between theaforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges”that extend from either end point of the range are specificallycontemplated. For example, a nested sub-range of an exemplary range of 1to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in onedirection, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the otherdirection.

As used herein, “reduces” means a negative alteration of at least 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

A “metabolite” refers to any substance produced by metabolism (e.g., agrowth by-product) or a substance necessary for taking part in aparticular metabolic process. Examples of metabolites include, but arenot limited to, enzymes, acids, solvents, gases, alcohols, proteins,vitamins, minerals, microelements, amino acids, biopolymers, andbiosurfactants.

As used herein, “surfactant” means a surface-active compound that lowersthe surface tension (or interfacial tension) between two liquids orbetween a liquid and a solid. Surfactants can act as, e.g., detergents,wetting agents, emulsifiers, foaming agents, and/or dispersants. A“biosurfactant” is a surfactant produced by a living cell.

The transitional term “comprising,” which is synonymous with“including,” or “containing,” is inclusive or open-ended and does notexclude additional, unrecited elements or method steps. By contrast, thetransitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim. The transitional phrase“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. Use of the term“comprising” contemplates other embodiments that “consist” or “consistessentially of” the recited component(s).

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “and” and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof. All references cited herein are hereby incorporated byreference in their entirety.

Compositions In preferred embodiments, the subject invention provides acomposition for improving oil and/or gas production, the compositioncomprising one or more solvents and one or more surfactants. In oneembodiment, any combination of solvents is utilized with any combinationof surfactants.

In one exemplary embodiment, the composition comprises one or more yeastfermentation products, one or more surfactants, one or more solvents,and one or more chelating agents. Optionally, one or more ammonium saltsand/or co-surfactants can also be included.

In one embodiment, the composition is customized based on the type ofparaffin that is being treated. For example, paraffin deposits can varyfrom soft accumulations to hard, brittle, solidified deposits. Thus, insome embodiments, the concentration of solvent in the composition can beincreased (e.g., up to about 10% of the composition) to boost thedispersal capabilities when harder paraffins are present. Accordingly,in certain embodiments, the practice of the subject invention comprisesobtaining an analyzing a sample of paraffin from the site to be treated.

Advantageously, the compositions is shelf stable for at least one weekor longer, and can be transported, stored and then applied selectivelyto an oil well at any point, for example, after a decline in oil and/orgas production is observed.

In one embodiment, the solvent(s) and/or the surfactant(s) can beproduced by non-biological means (e.g., chemical isolation, purificationand/or synthesis). In another embodiment, the solvents and/orsurfactants can be derived from natural or biological sources, such as,for example, the living cells of microorganisms, plants, fungi and/oranimals.

In one embodiment, the composition comprises a first yeast fermentationproduct that comprises a yeast strain and/or by-products produced duringcultivation of the yeast. In one embodiment, the microbe is a yeast orfungus, such as, for example, Wickerhamomyces anomalus (Pichia anomala),Starmerella bombicola or Meyerozyma guilliermondii (Pichiaguilliermondii). In certain embodiments, the yeasts are inactivated, forexample, using thermal inactivation, prior to being added to the subjectcomposition.

In certain embodiments, use of yeast fermentation products according tothe subject invention can be superior to, for example, purifiedmicrobial metabolites alone, due to, for example, the advantageousproperties of the yeast cell walls. These properties include highconcentrations of mannoprotein as a part of yeast cell wall's outersurface (mannoprotein is a highly effective bioemulsifier) and thepresence of biopolymer beta-glucan (also an effective emulsifier) inyeast cell walls. Additionally, the yeast fermentation product furthercan comprise biosurfactants capable of reducing both surface andinterfacial tension, enzymes capable of solubilizing heavy hydrocarbonand/or paraffinic compounds, and other metabolites (e.g., lactic acid,ethyl acetate, ethanol, etc.), in the culture.

In some embodiments, certain fungi, other than yeasts, have cell wallscontaining the same advantageous properties. Accordingly, fermentationproducts comprising non-yeast fungi can also be used according to thesubject invention.

In one embodiment, a first yeast fermentation product, designated as“Star 3+,” can be obtained via cultivation of a yeast, e.g.,Wickerhamomyces anomalus, using a modified form of solid statefermentation. The culture can be grown on a substrate with ample surfacearea onto which the yeasts can attach and propagate, such as, forexample, corn flour, rice, soybeans, chickpeas, pasta, oatmeal or beans.The culture can be washed out and used in liquid form, or blended withthe solid substrate, milled and/or micronized, and optionally, dried.This comprises the Star 3+ product. The product can be diluted in waterand/or brine fluids, for example, at least 5, 10, 100, 500 or 1,000times prior to being added to the composition.

In an alternative embodiment, the first yeast fermentation product isobtained using submerged fermentation, wherein the first yeastfermentation product comprises liquid broth and, optionally, cells andany yeast growth by-products resulting from the submerged fermentation.

The composition according to the subject invention can comprise one ormore solvents to aide in, for example, dissolving and dispersingparaffins. In one embodiment, a combination of solvents is utilized. Inone embodiment, the composition comprises solvents at a concentration ofabout 50% or less, 25% or less, or 10% or less, by volume.

Preferably, the one or more solvents are not produced by the yeasts ofthe yeast fermentation product, meaning they are included in addition toany solvents that may be produced by the yeast of the first yeastfermentation product.

Examples of solvent(s) that can be utilized according to the subjectinvention include, but are not limited to, terpenes, terpenoids,alcohols, ionic or semi-ionic liquids, acetates, aliphatic and/oraromatic hydrocarbons, olefins, esters, oxygenates, ketones, aceticacid, kerosene, gasoline, diesel, benzene, ethyl benzenes, propylbenzenes, butyl benzenes, toluene, ethyl toluenes, xylene, pentane,alkylene amines, dioxane, carbon disulfide, mesitylene, cumene, cymenes,saturated aliphatic and/or alicyclic hydrocarbons, naphtha, naphthenes,cyclohexane, decalin, tetralin, heptane, octane, cyclooctane, isooctane,cycloheptane, turpentine, carbon tetrachloride, ether alcohol, pinene,dialkyl ether and/or any combination thereof.

In one embodiment, the one or more solvents are non-polar aromaticsolvents. In one embodiment, the solvents can include one or more of,for example, terpenes, terpenoids, acetates, ionic or semi-ionicliquids, alcohols, kerosene, gasoline, diesel, benzene, toluene, and/orxylene.

In certain embodiments, the solvents can comprise one or more acetates.In one embodiment, the acetates are naturally-derived. In preferredembodiments, the acetates include isoamyl acetate and/or primary amylacetate. The acetate(s) can be included at a concentration of about 10ml/L to 200 ml/L, about 20 ml/L to 175 ml/L, about 30 ml/L to 150 ml/l,about 40 ml/L to 125 ml/L, or about 50 ml/L to 100 ml/L.

In certain embodiments, the solvents can comprise one or more terpenesand/or terpenoids. In some embodiments, the terpenes or terpenoids arederived from plants, such as citrus plants or pine trees. Terpenes andterpenoids can include but are not limited to, limonenes, orangeterpenes, lemon terpenes, grapefruit terpenes, orange oil, lemon oil,other citrus terpenes, other citrus oils, geraniol, terpineol,dipentene, myrcene, linalool, cymene and pinene.

In a preferred embodiment, the terpenes and/or terpenoids includeturpentine, D-limonene and/or dipentene at a concentration of about 1.0%to about 10.0% by weight, or about 2.0% to about 8.0% by weight. In oneembodiment, the concentration of turpentine, D-limonene and/or dipenteneis about 10 ml/L to 200 ml/L, about 20 ml/L to 175 ml/L, about 30 ml/Lto 150 ml/l, about 40 ml/L to 125 ml/L, or about 50 ml/L to 100 ml/L.

In certain embodiments, the solvents can comprise one or more alcohols,such as, for example, ethanol, methanol, propanol, isopropyl alcoholand/or hexanol. In one embodiment, the composition comprises hexanoland/or isopropyl alcohol, at a concentration of about 1 ml/L to 200ml/L, about 2 ml/L to 175 ml/L, about 3 ml/L to 150 ml/1, or about 4ml/L to 100 ml/L.

In certain embodiments, the solvents can comprise one or more ionic orsemi-ionic liquids. Exemplary ionic or semi-ionic liquids suitable forthe subject composition include, but are not limited to, ethyl ammoniumnitrate, and/or a semi-ionic mixture of glycerin/glycerol with magnesiumsulfate heptahydrate (MgSO₄.7H₂O). In one embodiment, the mixture ofglycerol and Epsom salt (MgSO₄.7H₂O) has a ratio of glycerol to Epsomsalt of 1:1 to 1:10, or from 1:1 to 10:1.

In some embodiments, the ionic or semi-ionic liquid can act as aco-solvent and can prevent the formation of ring bonds in hydrocarboncompositions, which is one cause of hydrocarbon precipitation.

In one embodiment, the ionic or semi-ionic liquid is present in thecomposition at a concentration of about 10 ml/L to 200 ml/L, about 20ml/L to 175 ml/L, about 30 ml/L to 150 ml/l, about 40 ml/L to 125 ml/L,or about 50 ml/L to 100 ml/L.

In one embodiment, the composition comprises one or more surfactants,which, along with paraffin removal and/or dispersal, can provideadditional enhanced oil recovery due to, for example, their surface andinterfacial tension reduction properties.

The surfactant(s) can be of non-biological origin and/or they can bebiosurfactants, meaning surfactants produced by a living cell.Non-biological surfactants can be selected from, for example, anionic,cationic, zwitterionic and/or nonionic classes of surfactants.

In certain embodiments, the surfactants are microbial biosurfactants ora blend of more than one type of biosurfactant. Biosurfactants are astructurally diverse group of surface-active substances produced bymicroorganisms. Biosurfactants are biodegradable and can produced usingselected organisms in or on renewable substrates.

All biosurfactants are amphiphiles. They consist of two parts: a polar(hydrophilic) moiety and non-polar (hydrophobic) group. Due to theiramphiphilic structure, biosurfactants increase the surface area ofhydrophobic water-insoluble substances, increase the waterbioavailability of such substances, and change the properties ofbacterial cell surfaces.

Furthermore, biosurfactants accumulate at interfaces, and reduce thesurface and interfacial tension between the molecules of liquids,solids, and gases, thus leading to the formation of aggregated micellarstructures in solution.

Biosurfactants according to the subject invention include, for example,low-molecular-weight glycolipids, lipopeptides, fatty acid estercompounds, fatty acid ether compounds, flavolipids, phospholipids, andhigh-molecular-weight polymers/biopolymers such as lipoproteins,lipopolysaccharide-protein complexes, and/orpolysaccharide-protein-fatty acid complexes. Preferably, thebiosurfactants are produced by microorganisms In one embodiment, thebiosurfactants can comprise one or more glycolipids such as, forexample, rhamnolipids, rhamnose-d-phospholipids, trehalose lipids,trehalose dimycolates, trehalose monomycolates, mannosylerythritollipids, cellobiose lipids, ustilagic acid and/or sophorolipids(including lactonic and/or acidic forms).

In an exemplary embodiment, the surfactant is a mannosylerythritol lipid(MEL), comprising either 4-O—B-D-mannopyranosyl-meso-erythritol or1-O—B-D-mannopyranosyl-meso-erythritol as the hydrophilic moiety, andfatty acid groups and/or acetyl groups as the hydrophobic moiety. One ortwo of the hydroxyls, typically at the C4 and/or C6 of the mannoseresidue, can be acetylated. Furthermore, there can be one to threeesterified fatty acids, from 8 to 12 carbons or more in chain length.

MEL molecules can be modified, either synthetically or in nature. Forexample, MEL can comprise different carbon-length chains or differentnumbers of acetyl and/or fatty acid groups.

MEL molecules and/or modified forms thereof according to the subjectinvention can include, for example, tri-acylated, di-acylated,mono-acylated, tri-acetylated, di-acetylated, mono-acetylated andnon-acetylated MEL, as well as stereoisomers and/or constitutionalisomers thereof.

In certain specific embodiments, the MEL molecules are selected frommembers of the following groups: MEL A (di-acetylated), MEL B(mono-acetylated at C4), MEL C (mono-acetylated at C6), MEL D(non-acetylated), tri-acetylated MEL A, tri-acetylated MEL B/C, andfurther including all possible isomers of the members of these groups.

Other MEL-like molecules that exhibit similar structures and similarproperties, can also be produced according to the subject invention,e.g., mannosyl-mannitol lipids (MML), mannosyl-arabitol lipids (MAL),and/or mannosyl-ribitol lipids (MRL).

In one embodiment, the biosurfactants can comprise one or morelipopeptides, such as, for example, surfactin, iturin, fengycin,arthrofactin, viscosin, amphisin, syringomycin, and/or lichenysin.

In one embodiment, the biosurfactants can comprise one or more othertypes of biosurfactants, such as, for example, cardiolipin, emulsan,lipomanan, alasan, and/or liposan.

In one embodiment, the surfactants can comprise one or moremicrobial-produced fatty acid ester compounds having physical propertiesand/or behaviors similar to those of biosurfactants, but which are notcommonly known as biosurfactants.

In certain embodiments, the fatty acid ester compounds can berepresented by the following formula:

wherein

-   -   Z═O    -   R₁═C₆ to C₂₂ saturated or unsaturated hydrocarbon, or an        epoxide, or cyclopropane thereof    -   Y₁═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₁    -   Y₂═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₁    -   Y₃═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₂    -   Y₄═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₂    -   R₂═C₁-C₁₀ saturated or unsaturated, branched or unbranched,        hydrocarbon.

In certain embodiments, the fatty acid ester compounds can include, forexample, highly esterified oleic fatty acids, such as oleic fatty acidethyl esters and/or oleic fatty acid methyl esters (FAME).

In one embodiment, the surfactants can comprise one or moremicrobial-produced fatty acid ether compounds having physical propertiesand/or behaviors similar to those of biosurfactants, but which are notcommonly known as biosurfactants.

In certain embodiments, the fatty acid ether compounds can berepresented by the following formula:

wherein

-   -   R₁═C₆ to C₂₂ saturated or unsaturated hydrocarbon, or an        epoxide, or cyclopropane thereof    -   Y₁═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₁    -   Y₂═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₁    -   Y₃═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₂    -   Y₄═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₂    -   R₂═C₁-C₁₀ saturated or unsaturated, branched or unbranched,        hydrocarbon.

In one embodiment, the biosurfactants can be added to the composition ina crude and/or purified form. In one embodiment, the concentration ofbiosurfactant is about 10 ml/L to 200 ml/L, about 25 ml/L to 175 ml/L,about 30 ml/L to 150 ml/l, about 40 ml/L to 125 ml/L, or about 50 ml/Lto 100 ml/L.

In preferred embodiments, the surfactant concentration is no lower thancritical micelle concentration (CMC) at the time the composition isintroduced into the formation (e.g., after natural dilution occurswithin the formation). Such concentration can be calculated by theskilled artisan having the benefit of the subject disclosure.

The biosurfactants can be present as a growth by-product of a cultivatedyeast, although preferably, they are included in addition to anybiosurfactants that may happen to be present as growth by-products inthe first yeast fermentation product.

In certain embodiments, the biosurfactant can be added to thecomposition in the form of a microbial culture, e.g., a second yeastfermentation product, containing liquid fermentation broth and cellsresulting from submerged cultivation of a biosurfactant-producingmicrobe, e.g., Wickerhamomyces anomalus, Starmerella bombicola orMeyerozyma guilliermondii. In certain embodiments, the second yeastfermentation is not produced using the same yeast as the first yeastfermentation product.

In a specific embodiment, when the biosurfactant is a sophorolipid(SLP), a second yeast fermentation product comprising fermentation brothwith Starmerella bombicola yeast cells and SLP therein, can be added tothe composition. The fermentation broth after, for example, 5 days ofcultivation at 25° C., can contain the yeast cell suspension and, forexample, 150 g/L or more of SLP.

The yeast cells may be active or inactive at the time they are added tothe composition. When lower concentrations of SLP are desired, the SLPportions of the culture, which forms a distinct layer in the culture,can be removed, and the residual liquid having, for example, 1-4 g/Lresidual SLP and, optionally, yeast cells and other growth by-products,can be utilized in the subject composition. When use of anotherbiosurfactant is desired, a similar product is envisioned that utilizesany other microbe capable of producing the other biosurfactant.

In one embodiment, the amount of the second yeast fermentation productin the composition is about 15 to 25% of the total composition byvolume, preferably about 20% of total volume.

In one embodiment, the surfactants of the compositions are obtainedthrough cultivation of microorganisms using processes ranging from smallto large scale. The cultivation process can be, for example, submergedcultivation, solid state fermentation (SSF), and/or a combinationthereof.

In one embodiment, the composition further comprises one or morechelating agents. As used herein, “chelator” or “chelating agent” meansan active agent capable of removing a metal ion from a system by forminga complex so that the metal ion, for example, cannot readily participatein or catalyze oxygen radical formation.

Examples of chelating agents suitable for the present invention include,but are not limited to, dimercaptosuccinic acid (DMSA),2,3-dimercaptopropanesulfonic acid (DMPS), alpha lipoic acid (ALA),thiamine tetrahydrofurfuryl disulfide (TTFD), penicillamine,ethylenediaminetetraacetic acid (EDTA), sodium acetate, sodium citrateand citric acid.

In one embodiment, the chelating agent is selected from EDTA, citricacid, citrate, sodium acetate, or a mixture thereof. The chelating agentor mixture thereof can be added to the composition in concentrations ofabout 1 g/L to about 50 g/L, or about 5 g/L to about 25 g/L, or about 10g/L to about 15 g/L. In specific embodiments, the chelating agent issodium citrate.

The subject composition can further comprise carriers (e.g., water, oiland/or brine fluids), as well as other optional compounds that areuseful for paraffin removal and/or enhanced oil recovery, such as, forexample, ammonium salts, co-surfactants, and/or enzymes. Theseadditional compounds can be added at concentrations ranging from, forexample, about 0.001% to 50%, about 1% to 25%, or about 10%, by weightor volume.

In one embodiment, the composition optionally comprises one or moreammonium salts, for example, ammonium hydroxide, ammonium phosphate,monoammonium phosphate, diammonium phosphate, ammonium chloride, oranother dibasic or monobasic ammonium salt. Advantageously, in oneembodiment, ammonium salts can serve pH adjusters in the composition,balancing the pH of the composition towards, or at, a neutral pH (e.g.,about pH 6 to 8) even in the presence of acidic substances, such asbrine fluids. This can be useful for improving the acid number of crudeoil recovered from the treated formation, as well as for preventing thecorrosion of equipment due to contact with acidic substances.

In some embodiments, the ammonium salt(s) comprise ammonium hydroxide(e.g., a 70% solution) at a concentration of about 1 ml/L to 10 ml/L, orabout 2 ml/L to 8 ml/L, or about 3 ml/L to 5 ml/L; and/or monoammoniumphosphate, at a concentration of about 1 g/L to 50 g/L, or about 2 g/Lto about 30 g/L, or about 10 g/L to about 20 g/L.

In one embodiment, the composition optionally comprises one or moreco-surfactants. In certain embodiments, the co-surfactant ismonoammonium phosphate or a surfactant as described previously herein,e.g., a MEL or an esterified fatty acid.

In certain embodiments, the composition comprises one or more yeastfermentation products, one or more surfactants, one or more solvents,one or more chelating agent(s), and, optionally, one or more ammoniumsalts and/or co-surfactants.

In one exemplary embodiment, the one or more yeast fermentation productscomprise Star 3+.

In one exemplary embodiment, the one or more surfactants comprise MEL,an esterified fatty acid, and/or SLP.

In one exemplary embodiment, the one or more solvents comprise isopropylalcohol, isoamyl acetate, primary amyl acetate, turpentine, dipenteneand/or D-limonene.

In one exemplary embodiment, the one or more chelating agents comprisesodium citrate.

In one exemplary embodiment, the optional one or more ammonium saltscomprise ammonium hydroxide and/or monoammonium phosphate. In certainembodiment, monoammonium phosphate can also serve as a co-surfactant.

In one embodiment, the composition can be diluted using water, oil, orany other diluent, including, for example, sterilized produced waterfrom an oil well.

Advantageously, the compositions of the subject invention provide a widerange of benefits to the oil and gas industry, including at all stagesof production. For example, the subject compositions can be used ascleaning agents to remove and liquefy paraffin deposits; paraffindispersants; emulsifiers; viscosity reducers; EOR agents; paraffininhibitors; antibacterial agents; corrosion inhibitors; and other uses,as are described throughout this description.

Further advantages to the subject compositions include that they can beformulated to be particularly potent for use in liquefying long-chainparaffins, which can be particularly difficult to dissolve;

they can be utilized in mature oil wells, or wells where hot oiling hasbeen implemented (these types of wells can contain deposits of complexparaffins, as well as deposits having greater thickness and/or soliditythan other wells);

they can be utilized in recovery and transport of oil in locations wherelower temperatures might cause paraffin deposition, such as, forexample, in offshore wells, in the arctic or Antarctic, and in climatesthat experience cold winter temperatures (e.g., as low as −32° C. orlower); and

they can be utilized in oil wells with high formation water salinitylevels (e.g., in geologic regions where formation water salinity is upto 250,000 ppm (total dissolved solids), up to 300,000 ppm, or even upto 400,000 ppm or more).

Methods for Treating Paraffin and Enhancing Oil Recovery

The subject invention provides materials and methods for improving oiland/or gas production from a well and/or a subterranean formation. Inparticular, the subject invention can be used to remove paraffins andother contaminants from wells, wellbores, subterranean formations andproduction equipment associated with wells, wellbores, and formations,that might, for example, obstruct or slow the flow of oil and/or gas.Furthermore, in one embodiment, the subject method can enhance oilrecovery from an oil well or formation.

The subject methods can be used in, for example, vertical, horizontaland/or fracking wells, mature wells, stripper (marginal) wells,flowlines, as well as to clean and/or maintain wellbores, piping,tubing, storage tanks, and other equipment. Advantageously, use of thesubject invention can improve and/or enhance oil recovery, aid in wellstimulation, and restore the health (e.g., production capacity) ofunder-producing or even dead wells.

In certain embodiments, the subject invention provides methods ofimproving oil and/or gas production, wherein a composition according tothe subject invention is applied to a subterranean formation, an oiland/or gas well, a wellbore, and/or equipment associated therewith. Incertain embodiments, the methods can also enhance oil recovery from theoil well. In some embodiments, the methods are implemented once the rateof oil production from a well has begun to decline due to, for example,obstructing contaminants.

Advantageously, the methods are effective at dissolving paraffinicbuildup without need for mechanical cleaning solutions. In someembodiments, the methods obviate the need for toxic solvents.

The methods can utilize compositions that are customized for aparticular well. Thus, in one embodiment, the method comprises testingthe well and/or associated equipment, analyzing the paraffin compositionpresent therein and determining the preferred formulation for thecomposition prior to treatment. Advantageously, the subject methods canbe useful for removal and dispersal of a broad spectrum of paraffintypes, including shorter chain paraffins as well as longer chainparaffins that are particularly difficult to remove due to, for example,the heaviness, thickness and/or the hardness of the deposit.

In one embodiment, the methods improve oil and/or natural gas productionthrough the removal and dispersal of paraffin deposits and/orprecipitates that have accumulated in a subterranean formation, in anoil and/or gas well, in a wellbore and/or in production equipmentassociated with any of these. For example, the methods are useful forremoving paraffin deposits from the rock pores of subterraneanformations, from wells, wellbores, and from equipment, such as, forexample, tubing, pipes, drills and tanks associated with all aspects ofoil and/or gas production. In certain embodiments, the subjectcompositions and methods can also free stuck or floating rods, allowinginoperable wells to resume operation. The methods can also be useful intreating mature wells and wells that have undergone hot oiling.

Additionally, the methods provide for recovery of economically valuableparaffin hydrocarbons by dispersing and/or emulsifying the dislodgedparaffin back into crude oil fluids. Advantageously, applying thesubject composition helps inhibit paraffin crystallization anddeposition, and helps prevent re-crystallization and re-deposition ofdispersed paraffins while pumping and transporting. The methods are eveneffective at keeping the paraffins suspended/emulsified in the crude oilfluids at temperatures less than 90° C., less than 50° C., less than 25°C., and even less than 0° C., for example from −3° C. to −32° C.

Furthermore, in addition to ultimately increasing the amounts of crudeoil recovered from a well due to the clearing and/or dispersing ofparaffin deposits, the methods also enhance oil recovery through, forexample, the amphiphilic properties of surfactants, includingbiosurfactants.

The subject methods can also be useful for a multitude of other benefitsrelated to oil and gas recovery, including, for example: inhibition ofparaffin crystallization and prevention of paraffin deposition;reduction in viscosity of paraffinic crude oil; reduction in pour pointof paraffinic crude oil (e.g., to about −25° F./−32° C.); removal and/ordissolution of scale; release of rust from oilfield casings and relatedequipment; protection against under-deposit rust-related corrosion ofequipment; inhibition of bacterial growth and disruption of biofilmformation on equipment; protection against microbial induced corrosion(MIC); alteration of the wettability of the near-wellbore surface towater-wet; and remediation of formation skin damage.

As used herein, “applying” a composition or product refers to contactingit with a target or site such that the composition or product can havean effect on that target or site. The effect can be due to, for example,the individual ingredients of the subject compositions and/or asynergistic combination thereof. There are multiple ways that the methodmay be implemented using a composition according to the subjectinvention, for example, the compositions can be injected into oil wellsand/or the piping, tubulars, casing, annulus, pumps, and tanksassociated with oil-bearing formations, oil wells, oil production, oiltransmission and oil transportation.

Application of the composition can be performed during drillingoperations (e.g., while drilling, while tripping-in or tripping-out ofthe hole, while circulating mud, while casing, while placing aproduction liner, and/or while cementing, etc.). Application can alsooccur as a production treatment, for example, by introducing thecomposition into an oil well after oil production is underway and/orafter a decline in the rate of oil production from the formation hasoccurred.

The volume of treatment used can be determined taking into account, forexample, formation porosity, permeability and deposit thickness. In someembodiments, the treatment can produce effects in less than 24 hours ofshut-in time.

In one exemplary embodiment, a composition of the subject invention ispoured or injected down the casing side (back lines) of a well andallowing it to mix with the fluid that is already in the well. Whenenough fluid is present, the composition can then optionally becirculated by, for example, a pump for 24-72 hours, preferably 48-72hours. Prior to circulating, the composition may be allowed to set for 8to 24 hours, for example. The setting time, circulating time and dosagedepend on the amount of paraffin and/or other contaminants anticipatedto be present, as well as the depth and size of the well. A basicinitial dosage can be, but is not limited to, 20 gallons of compositionand for maintaining a clear structure, at least about 5 gallons ofcomposition per well on periodic basis, e.g. biweekly, monthly,bimonthly.

In one exemplary embodiment, the methods comprise pumping, for example,100 to 1,000 gallons of more of the composition into and out of an oilwell. Injection rates can be determined by a skilled oil well operation,although, as an example, an injection rate of 1 to 20 gallons perminute, or 1 to 20 barrels per minute can be used in some embodiments.

In one exemplary embodiment, the methods comprise applying between about100-1,000 gallons, or 200 to 600 gallons of the composition into theannulus between the tubing and casing, where it can flow through thepump and into the tubing.

In some embodiments, the composition can be introduced into theformation through perforations in the casing. The composition may beforced into the surrounding formation by applied pressure or, if thecomposition is allowed to set at the bottom of the casing, thecomposition may seep into the formation without additional pressure. Thecomposition permeates the formation, improving the rate of oil recoveryby a number of mechanisms such as, for example, dissolving paraffin andother contaminant blockages in the formation pore throats.

The composition may be introduced by means of injection pumps intooff-shore gas or oil wells to reduce contaminants in well casings andtransmission lines. In addition to the problems associated with land oilwells, the lines and contents between the bottom of the ocean and theplatform associated with off-shore wells are cooled by the ocean or seawater, thus increasing the crystallization and deposition rate of scale,paraffin and asphaltene. To treat the lines, from 1-500 gallons up to1000 barrels, 10,000 barrels, or more, for example, of the compositioncan be introduced therein.

In some embodiments, brine fluids can be injected into a well after thesubject compositions in order to push the treatment deeper into theformation.

The subject treatment can be effective in a range of different geologicformations. For example, the subject invention can be useful informations as deep as about 7,000 feet or deeper, and as shallow asabout 1,500 feet or shallower. Additionally, the invention can be usefulin formations having a range of porosity and/or permeability, forexample from about 0.1% to about 20% or more. The invention can also beuseful in formations having a wide range of temperatures, pH, andsalinity.

In one embodiment, the subject methods can replace methods that utilizesynthetic or chemical paraffin inhibiters for preventingcrystallization, precipitation and/or deposition of paraffin.Furthermore, the subject methods can reduce or replace the need forphysical alteration of equipment to prevent paraffin crystallization anddeposition.

In one embodiment, the subject invention can be used to improve one ormore qualities of crude oil. For example, in one embodiment, the methodscan be used to reduce the viscosity of paraffinic crude oil, thusallowing for more efficient recovery of the oil from a well.

In another embodiment, improving one or more qualities of crude oilcomprises altering the pour point and/or cloud point of paraffinic crudeoil, for example, by lowering the pour point and/or cloud point.Reduction in cloud point and/or pour point allows for the methods andcomposition of the subject invention to be utilized in lowertemperatures, for example, with offshore oil wells, in formations andequipment present or being transported in colder climates, and/or duringthe winter. This is because, in the case of pour point, the temperatureat which the oil crystallizes and/or freezes is lower, and in the caseof cloud point, the temperature at which the dissolved solids andparaffins in the oil precipitate is lower. Thus, the subject inventioncan be used to prevent re-deposition of paraffins while pumping andtransport, even in colder temperatures. In a specific embodiment, thesubject invention can lower the pour point of paraffinic crude oil toabout −25° F., or to about −32° C.

In one embodiment, the subject methods can be used to remove and/ordissolve scale present in a formation and/or on equipment. Theseproblematic deposits can be formed by, for example, deposits ofprecipitated mineral salts, which can arise as a result of, for example,changes in the pressure, composition and/or temperature of the crudeoil. Scales can result from precipitates of, for example, bariumsulfate, calcium carbonate, strontium sulfate, calcium sulfate, sodiumchloride, silicon dioxide, iron sulfide, iron oxides, iron carbonate,silicates, phosphates and oxides, or any of a number of compounds thatare insoluble or mildly soluble in water.

In one embodiment, the methods of the subject invention can be used forpreventing corrosion associated with rust deposits, which can developunderneath paraffin deposits. In one embodiment, the compositions andmethods can also help release other rust deposits from oilfield casingsand other related equipment.

In one embodiment, the methods can be used to inhibit bacterial growthwithin an oil well or associated equipment, including inhibiting biofilmformation and/or disrupting biofilms present on the surfaces ofequipment. The invention can be useful against Gram-negative andGram-positive bacteria, such as chemoautotrophic bacteria,sulfate-reducing bacteria, sulfuric acid-producing bacteria,iron-oxidizing bacteria, and/or acid or ammonia-producing bacteria, andcan help protect oil and gas production equipment from MIC.

In one embodiment, the methods can open up channels and pores that areclogged with paraffin deposits, as well as with the adhesive/cohesivematrices that form when scale, polymers, sand, and other materialsbecome lodged in the paraffin, thus allowing for improved formationpermeability and oil production. In one embodiment, the subject methodscan also alter the wettability of formation rock so that it iswater-wet. Thus, the subject methods can be used to remediate formation“skin damage.”

Skin damage is an occurrence characterized by a zone of reducedpermeability within the vicinity of the wellbore. The reduction inpermeability can be a result of, for example, deposits, such asparaffins, asphaltenes, and bacterial biofilms, as well as alterationsin the wettability of formation rock from water-wet to oil-wet due to,for example, contaminating deposits, oil-based drilling fluids, and theuse of BTEX solvents.

The subject treatment can be effective in a range of different geologicformations. For example, the subject invention can be useful informations as deep as about 7,000 feet or deeper, and as shallow asabout 1,500 feet or shallower. Additionally, the invention can be usefulin formations having ranges of porosity and/or permeability, for examplefrom about 0.1% to about 20% or more.

The invention can also be useful in formations having a wide range oftemperatures, pH, and salinity. For example, the subject invention canbe utilized in recovery and transport of oil in locations where lowertemperatures might cause paraffin deposition, such as, for example, inoffshore wells, in the arctic or Antarctic, and in climates thatexperience cold winter temperatures.

Additionally, the subject invention can be utilized in oil wells withhigh formation water salinity levels. For example, the compositions canbe useful in geologic regions where formation water salinity is up to250,000 ppm (total dissolved solids), up to 300,000 ppm, or even up to400,000 ppm or more.

In certain embodiments, the methods can also be used for maintenance ofequipment, for example, pipes, tubulars, drills, pumps, casings, tanks,rods, boreholes, and other structures and equipment involved in oiland/or gas production and processing. In some embodiments, thecomposition may be applied directly to equipment. For example, prior toplacing rods and casings into gas and/or oil wells, these parts may besprayed with, or soaked in, the composition. The parts may be dippedinto tanks filled with the composition to prevent under-depositcorrosion and buildup of contaminants.

Any equipment or component of oil production, processing,transportation, storage and/or refining can be treated and maintainedwith a composition of the subject invention. Advantageously, the subjectinvention can be applied to equipment involved in all stages of thechain of operations, including exploration and production (E&P) (e.g.,onshore and offshore wellbores, flowlines, and tanks), midstream (e.g.,pipelines, tankers, transportation, storage tanks), and in refineries(e.g., heat exchangers, furnaces, distillation towers, cokers,hydrocrackers).

In one embodiment, maintenance of equipment is achieved through theprevention, removal, and/or dispersal of contaminating deposits thatform on the equipment. There are many types of contaminants associatedwith oil production equipment, such as paraffins, scales, oils,asphalts/asphaltenes, resins, sulfur, tar by-products, biofilms, andother viscous materials. The composition of the present invention can beused to remove any one or more of the contaminants associated with oilrecovery, transmission and processing. In certain specific embodiments,the contaminant is paraffin.

In one embodiment, the subject invention can be used for preventingprecipitation and/or deposition of contaminants from occurring. Thus,the present invention allows for delaying or completely removing thenecessity for preventative maintenance related to removing precipitatesand deposits, as well as the need for replacing or repairing equipmentparts.

The subject composition can further be applied for dissolving anddispersal of contaminant buildup in, for example, storage andtransportation tanks, tankers, ships, trucks, pipelines and flowlines,without need for mechanical cleaning solutions or toxic solvents.

In one embodiment, methods of cleaning a storage or transportation tankare provided, wherein air or methane is injected under pressure into atank. This can either be preceded by or followed by injection of thesubject composition. Waste water is pumped to a treatment plant aftertreatment with the subject composition. Preferably, the air or methaneis injected into the tank to allow for approximately 10 minutes ofroiling.

In certain embodiments of the subject methods, the composition may beapplied with a substance that promotes adherence of composition to asurface to be treated. The adherence-promoting substance may be acomponent of composition or it may be applied simultaneously with, orsequentially with, the composition. Adherence-promoters can includeorganic or inorganic particles, ions such as calcium, magnesium,phosphate, and sodium, iron, carbon sources that are metabolized toacetyl coenzyme A, acetyl phosphate, and acetate.

Up to, for example, 50 wt. % or more of further additives may beapplied, as needed, for particular applications, such as to vary the VOClevels, increase penetration of the composition, decrease viscosity ofthe composition, and/or as couplers for solvent insolubles in themixture.

Suitable additives include, but are not limited to, C8-C14 alcohol esterblends, glycols, glycol ethers, acid esters, diacid esters, petroleumhydrocarbons, amino acids, alkanolamines, amines, methyl or isobutylesters of C4-C6 aliphatic dibasic esters and n-methyl-2 pyrolidone.

C8-C14 alcohol ester blends include EXXATE 900, 1000, 1200 from ExxonChemical; glycols include propylene glycol, dipropylene glycol, andtriproplylene glycol; and glycol ethers include dipropylene glycolmonomethyl ether, propylene glycol monomethyl ether, propyleneglycol-n-butyl ether, ethylene glycol monobutyl ether, and diethyleneglycol monobutyl ether. Acid esters include methyl oleate and methyllinoleate, and diacid esters include methyl or butyl diesters ofglutaric, adipic, and succinic acids. Petroleum hydrocarbons includeAROMATIC 100, AROMATIC 150 ISOPAR M, and ISOPAR K.

Amines such as morpholine; 1,3-dimethyl-2-imidazolidinone; 1,3-propanediamine; 2-amino-1,3-propanediol; and 3-amino propanol; as wellas alkanolamines such as triethanolamine, diethanolamine, 2-aminomethylpropanol, and monoethanolamine act as dispersants for contaminants andsolubilize fatty acids and oils. Amino acids, provide nontoxicalternatives to monoethanolamine, and act as metal chelators. Methyl orisobutylesters of C4-C6 aliphatic dibasic esters and n-methyl-2pyrolidone are also useful.

Other additives typically used in compositions may be used, includingwater softening agents, sequesterants, corrosion inhibitors, andantioxidants, which are added in amounts effective to perform theirintended function. These additives and amounts thereof are well withinthe skill of the art. Suitable water softening agents include linearphosphates, styrene-maleic acid co-polymers, and polyacrylates. Suitablesequesterants include 1,3-dimethyl-2-immidazolidinone;1-phenyl-3-isoheptyl-1,3-propanedione; and 2hydroxy-5-nonylacetophenoneoxime. Examples of corrosion inhibitorsinclude 2-aminomethyl propanol, diethylethanolamine benzotraizole, andmethyl benzotriazole. Antioxidants suitable for the present inventioninclude (BHT) 2,6-di-tert-butyl-para-cresol, (BHA)2,6-di-tert-butyl-para-anisole, Eastman inhibitor O A BM-oxalyl bis(benzylidenehydrazide), and Eastman DTBMA 2,5-di-tert-butylhydroquinone.

All additives should have a flash point greater than 100° F., preferablygreater than 150° F. and more preferably 195° F. TCC in order to achievea final product flash point greater than 200° F.

In one embodiment, the subject methods can be used alongside and/or toenhance or supplement other methods of paraffin removal and/or enhancedoil recovery, e.g., other microbial, mechanical, thermal and/or chemicaltreatments.

The method can be used to replace dangerous high heat steaming or oilingmethods commonly used for paraffin removal. When, however, thermal,steaming and/or hot oil methods are used, the present method can be usedalongside (before, during or after) the thermal, steaming and/or hot oilto prevent recrystallization of the liquefied paraffins that aredispersed in the oil.

In one embodiment, the subject methods comprise a chemical treatment forremoving paraffin deposits present, for example, at or near a wellbore.The chemical treatment method can comprise applying a composition thatcomprises one or more chemical solvents and one or more non-biologicalsurfactants to the wellbore. Advantageously, the combination of solventswith surfactants, when compared to using, for example, solvents alone,can also provide enhanced oil recovery in addition to effectivelydispersing paraffin back into crude oil fluids. Additionally, thischemical treatment does not require large volumes of treatment mixture,as is required for treating deep into a subterranean formation. Thesurfactants can comprise, for example, from 1% to 50% of the volume ofthe chemical treatment composition, from 2% to 20%, or from 5% to 10%.

The subject compositions and methods can be used before and/or afteradministration of a mechanical, thermal and/or chemical treatment,and/or simultaneously therewith. Furthermore, the subject compositionsand methods can simply comprise a mechanical, thermal and/or chemicaltreatment on its own.

Examples of mechanical treatments include, but are not limited to,scraping, cutting and/or knifing, soluble pigs (made of, e.g.,naphthalene or microcrystalline wax) or insoluble pigs (made of, e.g.,plastic or hard rubber). Mechanical prevention of paraffin depositioncan include the use of plastic or coated pipes, or other low-friction,smooth surfaces on equipment.

Examples of thermal treatments include, but are not limited to,steaming, hot watering and/or hot oiling.

Examples of chemical paraffin treatments include, but are not limitedto, non-biological (e.g., produced by chemical purification, isolation,and/or synthesis) surfactants, condensates, solvents and/or inhibitors.

Surfactants are surface active agents having two functional groups,namely a hydrophilic (water-soluble) or polar group and a hydrophobic(oil-soluble) or non-polar group. The hydrophobic group is usually along hydrocarbon chain (C8-C18), which may or may not be branched, whilethe hydrophilic group is formed by moieties such as carboxylates,sulfates, sulfonates (anionic), alcohols, polyoxyethylenated chains(nonionic) and quaternary ammonium salts (cationic).

Non-biological surfactants according to the subject compositions andmethods include, but are not limited to: anionic surfactants, ammoniumlauryl sulfate, sodium lauryl sulfate (also called SDS, sodium dodecylsulfate), alkyl-ether sulfates sodium laureth sulfate (also known assodium lauryl ether sulfate (SLES)), sodium myreth sulfate; docusates,dioctyl sodium sulfosuccinate, perfluorooctanesulfonate (PFOS),perfluorobutanesulfonate, linear alkylbenzene sulfonates (LABs),alkyl-aryl ether phosphates, alkyl ether phosphate; carboxylates, alkylcarboxylates (soaps), sodium stearate, sodium lauroyl sarcosinate,carboxylate-based fluorosurfactants, perfluorononanoate,perfluorooctanoate; cationic surfactants, pH-dependent primary,secondary, or tertiary amines, octenidine dihydrochloride, permanentlycharged quaternary ammonium cations, alkyltrimethylammonium salts, cetyltrimethylammonium bromide (CTAB) (a.k.a. hexadecyl trimethyl ammoniumbromide), cetyl trimethylammonium chloride (CTAC), cetylpyridiniumchloride (CPC), benzalkonium chloride (BAC), benzethonium chloride(BZT), 5-Bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammoniumchloride, cetrimonium bromide, dioctadecyldi-methylammonium bromide(DODAB); zwitterionic (amphoteric) surfactants, sultaines CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate),cocamidopropyl hydroxysultaine, betaines, cocamidopropyl betaine,phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine,sphingomyelins; nonionic surfactants, ethoxylate, long chain alcohols,fatty alcohols, cetyl alcohol, stearyl alcohol, cetostearyl alcohol,oleyl alcohol, polyoxyethylene glycol alkyl ethers (Brij):CH3-(CH2)10-16-(O—C2H4)1-25-OH (octaethylene glycol monododecyl ether,pentaethylene glycol monododecyl ether), polyoxypropylene glycol alkylethers: CH3-(CH2)10-16-(0-C3H6)1-25-OH, glucoside alkyl ethers:CH3-(CH2)10-16-(0-Glucoside)1-3-OH (decyl glucoside, lauryl glucoside,octyl glucoside), polyoxyethylene glycol octylphenol ethers:C8H17-(C6H4)-(O—C2H4)1-25-OH (Triton X-100), polyoxyethylene glycolalkylphenol ethers: C9H19-(C6H4)O—(—C2H4)1-25-OH (nonoxynol-9), glycerolalkyl esters (glyceryl laurate), polyoxyethylene glycol sorbitan alkylesters (polysorbate), sorbitan alkyl esters (spans), cocamide MEA,cocamide DEA, dodecyldimethylamine oxide, copolymers of polyethyleneglycol and polypropylene glycol (poloxamers), and polyethoxylated tallowamine (POEA).

Anionic surfactants contain anionic functional groups at their head,such as sulfate, sulfonate, phosphate, and carboxylates. Prominent alkylsulfates include ammonium lauryl sulfate, sodium lauryl sulfate (alsocalled SDS, sodium dodecyl sulfate) and the related alkyl-ether sulfatessodium laureth sulfate, also known as sodium lauryl ether sulfate(SLES), and sodium myreth sulfate. Carboxylates are the most commonsurfactants and comprise the alkyl carboxylates (soaps), such as sodiumstearate.

Surfactants with cationic head groups include: pH-dependent primary,secondary, or tertiary amines; octenidine dihydrochloride; permanentlycharged quaternary ammonium cations such as alkyltrimethylammoniumsalts: cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethylammonium bromide, cetyl trimethylammonium chloride (CTAC);cetylpyridinium chloride (CPC); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; cetrimonium bromide; anddioctadecyldi-methylammonium bromide (DODAB).

Zwitterionic (amphoteric) surfactants have both cationic and anioniccenters attached to the same molecule. The cationic part is based onprimary, secondary, or tertiary amines or quaternary ammonium cations.The anionic part can be more variable and include sulfonates.Zwitterionic surfactants commonly have a phosphate anion with an amineor ammonium, such as is found in the phospholipids phosphatidylserine,phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins.

A surfactant with a non-charged hydrophilic part, e.g. ethoxylate, isnon-ionic. Many long chain alcohols exhibit some surfactant properties.

Condensates are low-density mixtures of hydrocarbon liquids present asgaseous components in raw natural gas that will condense to liquid statedepending on decrease in temperature and changes in pressure. Gascondensates generally comprise propane, butane, pentane, hexane, andother compounds. Condensates can be used as chemical treatments,including as solvents, in paraffin removal in oil and gas wells andequipment.

Examples of solvents and/or condensates according to the subjectcompositions and methods include, but are not limited to, aliphaticand/or terpenes, terpenoids, acetates, ionic liquids, alcohols, aromatichydrocarbons, ketones, acetic acid, kerosene, gasoline, diesel, benzene,ethyl benzenes, propyl benzenes, butyl benzenes, toluene, ethyltoluenes, xylene, pentane, alkylene amines, dioxane, carbon disulfide,mesitylene, cumene, cymenes, saturated aliphatic and/or alicyclichydrocarbons, naphtha, naphthenes, cyclohexane, decalin, tetralin,heptane, octane, cyclooctane, isooctane, cycloheptane, turpentine,carbon tetrachloride, ether alcohol, pinene, dialkyl ether and/or anycombination thereof.

In one embodiment, the subject methods can be utilized alongside and/orin combination with enzyme treatments for hydrocarbon deposit removaland/or enhanced oil recovery. Enzymes are typically divided into sixclasses: oxidoreductases, transferases, hydrolases, lyases, isomerasesand ligases. Each class is further divided into subclasses and byaction. Specific subclasses of enzymes according to the subjectinvention include, but are not limited to, proteases, amylases,glycosidases, cellulases, glucosidases, glucanases, galactosidases,moannosidases, sucrases, dextranases, hydrolases, methyltransferases,phosphorylases, dehydrogenases (e.g., glucose dehydrogenase, alcoholdehydrogenase), oxygenases (e.g., alkane oxygenases, methanemonooxygenases, dioxygenases), hydroxylases (e.g., alkane hydroxylase),esterases, lipases, ligninases, mannanases, oxidases, laccases,tyrosinases, cytochrome P450 enzymes, peroxidases (e.g.,chloroperoxidase and other haloperoxidasese), lactases, extracellularenzymes from Aspergillus spp. and other microbial species (e.g., lipasesfrom Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, Serratiamarcescens, Pseudomonas aeruginosa, and Staphylococcus aureus) and otherenzyme-based products known in the oil and gas industry.

Production of Microorganisms

The subject invention provides methods for cultivation of microorganismsand production of microbial metabolites and/or other by-products ofmicrobial growth. In one embodiment, the subject invention providesmaterials and methods for the production of biomass (e.g., viablecellular material), extracellular metabolites (e.g. small molecules andexcreted proteins), residual nutrients and/or intracellular components(e.g. enzymes and other proteins).

In certain embodiments, a microbe growth facility produces fresh,high-density microorganisms and/or microbial growth by-products ofinterest on a desired scale. The microbe growth facility may be locatedat or near the site of application, or at a different location. Thefacility produces high-density microbe-based compositions in batch,quasi-continuous, or continuous cultivation.

In certain embodiments, the microbe growth facilities of the subjectinvention can be located at or near the location where the microbe-basedproduct will be used (e.g., at or near an oil well) For example, themicrobe growth facility may be less than 300, 250, 200, 150, 100, 75,50, 25, 15, 10, 5, 3, or 1 mile from the location of use.

The microbe growth facilities can produce fresh, microbe-basedcompositions, comprising the microbes themselves, microbial metabolites,and/or other components of the medium in which the microbes are grown.If desired, the compositions can have a high density of vegetative cellsor a mixture of vegetative cells, spores, conidia, mycelia and/or othermicrobial propagules. Advantageously, the compositions can be tailoredfor use at a specified location. In one embodiment, the microbe growthfacility is located on, or near, a site where the microbe-based productswill be used.

Advantageously, in preferred embodiments, the methods of the subjectinvention harness the power of naturally-occurring local microorganismsand their metabolic by-products to improve oil production, transmissionand/or refining. Local microbes can be identified based on, for example,salt tolerance, ability to grow at high temperatures, and the use ofgenetic identification of the sequences described herein.

The microbe growth facilities provide manufacturing versatility by theirability to tailor the microbe-based products to improve synergies withdestination geographies. The microbe growth facilities may operate offthe grid by utilizing, for example, solar, wind and/or hydroelectricpower. Thus, the microbe-based compositions can be produced in remotelocations.

The growth vessel used for growing microorganisms can be any fermenteror cultivation reactor for industrial use. In one embodiment, the vesselmay have functional controls/sensors or may be connected to functionalcontrols/sensors to measure important factors in the cultivationprocess, such as pH, oxygen, pressure, temperature, agitator shaftpower, humidity, viscosity and/or microbial density and/or metaboliteconcentration.

In a further embodiment, the vessel may also be able to monitor thegrowth of microorganisms inside the vessel (e.g., measurement of cellnumber and growth phases). Alternatively, a daily sample may be takenfrom the vessel and subjected to enumeration by techniques known in theart, such as dilution plating technique. Dilution plating is a simpletechnique used to estimate the number of microbes in a sample. Thetechnique can also provide an index by which different environments ortreatments can be compared.

In one embodiment, the cultivation utilizes a medium supplemented with anitrogen source. The nitrogen source can be, for example, potassiumnitrate, ammonium nitrate ammonium sulfate, ammonium phosphate, ammonia,urea, and/or ammonium chloride. These nitrogen sources may be usedindependently or in a combination of two or more.

In one embodiment, the cultivation supplies oxygenation to the growingculture. One embodiment utilizes slow motion of air to remove low-oxygencontaining air and introduce oxygenated air. In the case of submergedfermentation, the oxygenated air may be ambient air supplemented dailythrough mechanisms including impellers for mechanical agitation of theliquid, and air spargers for supplying bubbles of gas to the liquid fordissolution of oxygen into the liquid.

In one embodiment, the cultivation utilizes a medium supplemented with acarbon source. The carbon source is typically a carbohydrate, such asglucose, sucrose, lactose, fructose, trehalose, mannose, mannitol,and/or maltose; organic acids such as acetic acid, fumaric acid, citricacid, propionic acid, malic acid, malonic acid, and/or pyruvic acid;alcohols such as ethanol, isopropyl, propanol, butanol, pentanol,hexanol, isobutanol, and/or glycerol; fats and oils such as soybean oil,rice bran oil, canola oil, olive oil, corn oil, sesame oil, and/orlinseed oil; etc. These carbon sources may be used independently or in acombination of two or more.

In one embodiment, the method comprises use of two carbon sources, oneof which is a saturated oil selected from canola, vegetable, corn,coconut, olive, or any other oil suitable for use in, for example,cooking. In a specific embodiment, the saturated oil is 15% canola oilor discarded oil that has been used for cooking.

In one embodiment, the microorganisms can be grown on a solid orsemi-solid substrate, such as, for example, corn, wheat, soybean,chickpeas, beans, oatmeal, pasta, rice, and/or flours or meals of any ofthese or other similar substances.

In one embodiment, growth factors and trace nutrients for microorganismsare included in the medium. This is particularly preferred when growingmicrobes that are incapable of producing all of the vitamins theyrequire. Inorganic nutrients, including trace elements such as iron,zinc, copper, manganese, molybdenum and/or cobalt may also be includedin the medium. Furthermore, sources of vitamins, essential amino acids,and microelements can be included, for example, in the form of flours ormeals, such as corn flour, or in the form of extracts, such as yeastextract, potato extract, beef extract, soybean extract, banana peelextract, and the like, or in purified forms. Amino acids such as, forexample, those useful for biosynthesis of proteins, can also beincluded.

In one embodiment, inorganic salts may also be included. Usableinorganic salts can be potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate,magnesium chloride, iron sulfate, iron chloride, manganese sulfate,manganese chloride, zinc sulfate, lead chloride, copper sulfate, calciumchloride, calcium carbonate, sodium chloride and/or sodium carbonate.These inorganic salts may be used independently or in a combination oftwo or more.

In some embodiments, the method for cultivation may further compriseadding additional acids and/or antimicrobials in the liquid mediumbefore and/or during the cultivation process. Antimicrobial agents orantibiotics are used for protecting the culture against contamination.Additionally, antifoaming agents may also be added to prevent theformation and/or accumulation of foam during cultivation.

The pH of the mixture should be suitable for the microorganism ofinterest. Buffers, and pH regulators, such as carbonates and phosphates,may be used to stabilize pH near a preferred value. When metal ions arepresent in high concentrations, use of a chelating agent in the liquidmedium may be necessary.

In one embodiment, the method for cultivation of microorganisms iscarried out at about 50 to about 100° C., preferably, 15 to 60° C., morepreferably, 25 to 50° C. In a further embodiment, the cultivation may becarried out continuously at a constant temperature. In anotherembodiment, the cultivation may be subject to changing temperatures.

In one embodiment, the equipment used in the method and cultivationprocess is sterile. The cultivation equipment such as the reactor/vesselmay be separated from, but connected to, a sterilizing unit, e.g., anautoclave. The cultivation equipment may also have a sterilizing unitthat sterilizes in situ before starting the inoculation. Air can besterilized by methods know in the art. For example, the ambient air canpass through at least one filter before being introduced into thevessel. In other embodiments, the medium may be pasteurized or,optionally, no heat at all added, where the use of low water activityand low pH may be exploited to control undesirable bacterial growth.

In one embodiment, the subject invention provides methods of producing amicrobial metabolite by cultivating a microbe strain of the subjectinvention under conditions appropriate for growth and production of themetabolite; and, optionally, purifying the metabolite. In a specificembodiment, the metabolite is a biosurfactant. The metabolite may alsobe, for example, solvents, acids, ethanol, lactic acid, manno-proteins,beta-glucan, proteins, amino acids, peptides, metabolic intermediates,polyunsaturated fatty acids, and lipids. The metabolite content producedby the method can be, for example, at least 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%.

The biomass content of the fermentation medium may be, for example from5 g/l to 180 g/l or more, or from 10 g/i to 150 g/l.

The microbial growth by-product produced by microorganisms of interestmay be retained in the microorganisms or secreted into the growthmedium. In another embodiment, the method for producing microbial growthby-product may further comprise steps of concentrating and purifying themicrobial growth by-product of interest. In a further embodiment, themedium may contain compounds that stabilize the activity of microbialgrowth by-product.

The method for cultivation of microorganisms and production of themicrobial by-products can be performed in a batch, quasi-continuous, orcontinuous processes.

In one embodiment, all of the microbial cultivation composition isremoved upon the completion of the cultivation (e.g., upon, for example,achieving a desired cell density, or density of a specified metabolite).In this batch procedure, an entirely new batch is initiated uponharvesting of the first batch.

In another embodiment, only a portion of the fermentation product isremoved at any one time. In this embodiment, biomass with viable cellsremains in the vessel as an inoculant for a new cultivation batch. Thecomposition that is removed can be a microbe-free medium or containcells, spores, mycelia, conidia or other microbial propagules. In thismanner, a quasi-continuous system is created.

Advantageously, the methods of cultivation do not require complicatedequipment or high energy consumption. The microorganisms of interest canbe cultivated at small or large scale on site and utilized, even beingstill-mixed with their media. Similarly, the microbial metabolites canalso be produced at large quantities at the site of need.

Because, in certain embodiments, the microbe-based products can begenerated locally, without resort to the microorganism stabilization,preservation, storage and transportation processes of conventionalmicrobial production, a much higher density of live microbes, spores,mycelia, conidia or other microbial propagules can be generated, therebyrequiring a smaller volume of the microbe-based product for use in theon-site application or which allows much higher density microbialapplications where necessary to achieve the desired efficacy. Thisallows for a scaled-down bioreactor (e.g., smaller fermentation tank,smaller supplies of starter material, nutrients and pH control agents),which makes the system efficient. Local generation of the microbe-basedproduct also facilitates the inclusion of the growth medium in theproduct. The medium can contain agents produced during the fermentationthat are particularly well-suited for local use.

Locally-produced high density, robust cultures of microbes are moreeffective in the field than those that have undergone vegetative cellstabilization, have been sporulated or have sat in the supply chain forsome time. The microbe-based products of the subject invention areparticularly advantageous compared to traditional products whereincells, spores, mycelia, conidia and/or other microbial propagules havebeen separated from metabolites and nutrients present in thefermentation growth media. Reduced transportation times allow for theproduction and delivery of fresh batches of microbes and/or theirmetabolites at the time and volume as required by local demand.

Advantageously, local microbe growth facilities provide a solution tothe current problem of relying on far-flung industrial-sized producerswhose product quality suffers due to upstream processing delays, supplychain bottlenecks, improper storage, and other contingencies thatinhibit the timely delivery and application of, for example, a viable,high cell- and/or propagule-count product and the associated growthmedium and metabolites in which the microbes are originally grown.

Local production and delivery within, for example, 24 hours offermentation results in pure, high cell density compositions andsubstantially lower shipping costs. Given the prospects for rapidadvancement in the development of more effective and powerful microbialinoculants, consumers will benefit greatly from this ability to rapidlydeliver microbe-based products.

Preparation of Microbe-Based Products

The subject invention provides microbe-based products (e.g., yeastfermentation products) for use in removing contaminants (e.g., paraffin)from oil wells, oil production equipment, and subterranean formations.One microbe-based product of the subject invention is simply thefermentation medium containing the microorganism and/or the microbialmetabolites produced by the microorganism and/or any residual nutrients.The product of fermentation may be used directly without extraction orpurification. If desired, extraction and purification can be easilyachieved using standard extraction and/or purification methods ortechniques described in the literature.

In one embodiment, a first yeast fermentation product, designated as“Star 3+,” can be obtained via cultivation of a yeast, e.g.,Wickerhamomyces anomalus, using a modified form of solid statefermentation. The culture can be grown on a substrate with ample surfacearea onto which the yeasts can attach and propagate, such as, forexample, rice, soybeans, chickpeas, pasta, oatmeal or beans. The entirefermentation medium with yeast cells growing throughout, and any growthby-products thereof (e.g., enzymes, solvents, and/or biosurfactants),can be harvested after, for example, 3-5 days of cultivation at 25-30°C. The culture can be blended with the substrate, milled and/ormicronized, and optionally, dried. This comprises the Star 3+ product.The composition, which can comprise 10¹⁰ to 10¹² cells/gram, can bediluted, for example, up to 10, 50, 100, 500, or 1,000 times prior tobeing mixed with other components.

In an alternative embodiment, the first yeast fermentation product isobtained using submerged fermentation, wherein the yeast fermentationproduct comprises liquid broth comprising cells and any yeast growthby-products. A liquid medium containing necessary sources of carbon,nitrogen, minerals and optionally, antimicrobial substances to preventcontaminating bacterial growth can be used. The culture can be grownwith an additional carbon source, particularly, a saturated oil (e.g.,15% canola oil, or used cooking vegetable oil). Typically, the pH beginsat 5.0-5.5, then decreases to 3.0-3.5, where it is stabilized. Thefermentation broth with cells and yeast growth by-products, which can beharvested after, for example, 24-72 hours of cultivation at 25-30° C.,comprises this alternative form of the Star 3+ product.

In one embodiment, a second yeast fermentation product can be obtainedvia submerged cultivation of a biosurfactant-producing yeast, e.g.,Starmerella bombicola. This yeast is an effective producer of glycolipidbiosurfactants, such as SLP. The fermentation broth after 5 days ofcultivation at 25° C. can contain the yeast cell suspension and, forexample, 150 g/L or more of SLP.

The second yeast fermentation can be further modified if lessbiosurfactant is desired in the composition. For example, fermentationof S. bombicola results in separation of the SLP into a distinguishablelayer. This SLP layer can be removed and the residual liquid andbiomass, which can still contain 1-4 g/L of residual SLP, can then beutilized a in the subject composition.

The microorganisms in the microbe-based product may be in an active orinactive form. In preferred embodiments, the microbes are inactivatedprior to adding to the compositions of the subject invention.

The microbe-based products may be used without further stabilization,preservation, and storage. Advantageously, direct usage of thesemicrobe-based products preserves a high viability of the microorganismsup until inactivation, reduces the possibility of contamination fromforeign agents and undesirable microorganisms, and maintains theactivity of the by-products of microbial growth.

The microbes and/or medium (e.g., broth or solid substrate) resultingfrom the microbial growth can be removed from the growth vessel andtransferred via, for example, piping for immediate use.

In one embodiment, the microbe-based product is simply the growthby-products of the microorganism. For example, biosurfactants producedby a microorganism can be collected from a submerged fermentation vesselin crude form, comprising, for example about 0.001% to about 99% purebiosurfactant in liquid broth.

In other embodiments, the microbe-based product (microbes, medium, ormicrobes and medium) can be placed in containers of appropriate size,taking into consideration, for example, the intended use, thecontemplated method of application, the size of the fermentation vessel,and any mode of transportation from microbe growth facility to thelocation of use. Thus, the containers into which the microbe-basedcomposition is placed may be, for example, from 1 gallon to 1,000gallons or more. In other embodiments the containers are 2 gallons, 5gallons, 25 gallons, or larger.

In one embodiment, the yeast fermentation product according to thesubject composition comprises a yeast strain and/or growth by-productsthereof.

In certain embodiments, use of yeast fermentation products according tothe subject invention can be superior to, for example, purifiedmicrobial metabolites alone, due to, for example, the advantageousproperties of the yeast cell walls. These properties include highconcentrations of mannoprotein as a part of yeast cell wall's outersurface (mannoprotein is a highly effective bioemulsifier) and thepresence of biopolymer beta-glucan (an emulsifier) in yeast cell walls.Additionally, the yeast fermentation product further can comprisebiosurfactants in the culture, which are capable of reducing bothsurface and interfacial tension, and other metabolites (e.g., lacticacid, ethyl acetate, ethanol, etc.) in the culture.

Upon harvesting, for example, the yeast fermentation product, from thegrowth vessels, further components can be added as the harvested productis placed into containers and/or piped (or otherwise transported foruse). The additives can be, for example, buffers, carriers, othermicrobe-based compositions produced at the same or different facility,viscosity modifiers, preservatives, nutrients for microbe growth,tracking agents, solvents, biocides, other microbes and otheringredients specific for an intended use.

Other suitable additives, which may be contained in the formulationsaccording to the invention, include substances that are customarily usedfor such preparations. Examples of such additives include surfactants,emulsifying agents, lubricants, buffering agents, solubility controllingagents, pH adjusting agents, preservatives, stabilizers and ultra-violetlight resistant agents.

In one embodiment, the product may further comprise buffering agentsincluding organic and amino acids or their salts. Suitable buffersinclude citrate, gluconate, tartarate, malate, acetate, lactate,oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate,glucarate, tartronate, glutamate, glycine, lysine, glutamine,methionine, cysteine, arginine and a mixture thereof. Phosphoric andphosphorous acids or their salts may also be used. Synthetic buffers aresuitable to be used but it is preferable to use natural buffers such asorganic and amino acids or their salts listed above.

In a further embodiment, pH adjusting agents include potassiumhydroxide, ammonium hydroxide, potassium carbonate or bicarbonate,hydrochloric acid, nitric acid, sulfuric acid or a mixture.

In one embodiment, additional components such as an aqueous preparationof a salt such as sodium bicarbonate or carbonate, sodium sulfate,sodium phosphate, sodium biphosphate, can be included in theformulation.

Advantageously, in accordance with the subject invention, themicrobe-based product may comprise medium in which the microbes weregrown. The product may be, for example, at least, by weight, 1%, 5%,10%, 25%, 50%, 75%, or 100% growth medium. The amount of biomass in theproduct, by weight, may be, for example, anywhere from 0% to 100%, 10%to 90%, 20% to 80%, or 30% to 70%, inclusive of all percentagestherebetween.

Optionally, the product can be stored prior to use. The storage time ispreferably short. Thus, the storage time may be less than 60 days, 45days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2days, 1 day, or 12 hours. In a preferred embodiment, if live cells arepresent in the product, the product is stored at a cool temperature suchas, for example, less than 20° C., 15° C., 10° C., or 5° C. On the otherhand, a biosurfactant composition can typically be stored at ambienttemperatures.

Advantageously, the subject products can be used to simultaneouslyenhance oil recovery (e.g., by stimulating an oil well), while removingparaffin and other contaminants from oil production equipment andoil-bearing formations.

Microbial Strains

The microorganisms useful according to the subject invention can be, forexample, bacteria, yeast and/or fungi. These microorganisms may benatural, or genetically modified microorganisms. For example, themicroorganisms may be transformed with specific genes to exhibitspecific characteristics. The microorganisms may also be mutants of adesired strain. As used herein, “mutant” means a strain, genetic variantor subtype of a reference microorganism, wherein the mutant has one ormore genetic variations (e.g., a point mutation, missense mutation,nonsense mutation, deletion, duplication, frameshift mutation or repeatexpansion) as compared to the reference microorganism. Procedures formaking mutants are well known in the microbiological art. For example,UV mutagenesis and nitrosoguanidine are used extensively toward thisend.

In preferred embodiments, the microorganism is any yeast or fungus,including, for example, Acaulospora, Aspergillus, Aureobasidium (e.g.,A. pullulans), Blakeslea, Candida (e.g., C. albicans, C. apicola),Debaryomyces (e.g., D. hansenii), Entomophthora, Fusarium, Hanseniaspora(e.g., H. uvarum), Hansenula, Issatchenkia, Kluyveromyces, Mortierella,Mucor (e.g., M. piriformis), Penicillium, Phythium, Phycomyces, Pichia(e.g., P. anomala, P. guielliermondii, P. occidentalis, P.kudriavzevii), Pseudozyma (e.g., P. aphidis), Rhizopus, Saccharomyces(S. cerevisiae, S. boulardii sequela, S. torula), Starmerella (e.g., S.bombicola), Torulopsis, Thraustochytrium, Trichoderma (e.g., T. reesei,T. harzianum, T. virens), Ustilago (e.g., U. maydis), Wickerhamomyces(e.g., W. anomalus), Williopsis, and/or Zygosaccharomyces (e.g., Z.bailii).

In one embodiment, the microbial strain is a Pichia yeast, or a relatedspecies selected from Wickerhamomyces anomalus (Pichia anomala),Meyerozyma guilliermondii (Pichia guilliermondii) and Pichiakudriavzevii. In one embodiment, the yeast or fungus is Starmerellabombicola, Pseudozyma aphidis, or Saccharomyces cerevisiae.

In one embodiment, the yeast is Wickerhamomyces anomalus. W. anomalusproduces a killer toxin comprising exo-β-1,3-glucanase. Additionally, W.anomalus produces biosurfactants that are capable of reducingsurface/interfacial tension of water, as well as various other usefulsolvents, enzymes and other metabolites, such as, for example, phytase,glycosidases, ethyl acetate, acetic acid, lactic acid, and ethanol.

In one embodiment, the yeast is Starmerella bombicola, which is aneffective producer of, for example, glycolipid biosurfactants.

In one embodiment, the yeast is Meyerozyma guilliermondii, which is aneffective producer of, for example, glycolipid biosurfactants and/oresterified fatty acid compounds.

Other microbial strains can be used in accordance with the subjectinvention, including, for example, any other yeast and/or fungal strainshaving high concentrations of mannoprotein and/or beta-glucan in theircell walls and/or that are capable of producing biosurfactants and othermetabolites such as, e.g., lactic acid, ethyl acetate and ethanol.

EXAMPLES

A greater understanding of the present invention and of its manyadvantages may be had from the following examples, given by way ofillustration. The following examples are illustrative of some of themethods, applications, embodiments and variants of the presentinvention. They are not to be considered as limiting the invention.Numerous changes and modifications can be made with respect to theinvention.

Example 1—Cultivation of First Yeast Fermentation Product

The first yeast fermentation product according to the subject invention,designated as “Star 3+,” can be obtained via cultivation of, e.g.,Wickerhamomyces anomalus, using a solid or semi-solid substrate.

For example, corn flour, chickpeas, soybeans, rice or other similarfoodstuff items can be mixed with nutrient medium seeded with, forexample, 1×10¹² cells/ml of W. anomalus. After about 3-5 days of growthat about 25-35° C., preferably, 28° C., the yeast and any growthby-products thereof (e.g., enzymes, solvents, and/or biosurfactants) caneither be washed out and utilized in liquid form, or the yeast andsubstrate can be milled, blended and/or micronized, and optionallydried. The yeasts can be inactivated by, for example, thermal means.

The product can be diluted by 10-1,000 times prior to mixing with othercomposition ingredients. When more than one strain of microorganism isutilized in the composition, the different strains of microbe areproduced and stored separately and then mixed together prior to use.

Example 2—Crude Oil Recovery from Aged Sand

Aged sand used for crude oil recovery is prepared by mixing sand withcrude oil and “aging” the mixture for 3 days at 60° C. Six test tubesare filled with the mixture. The crude oil content in the sand is 18%,or an estimated 3.6 ml per tube. Each tube is treated with 25 mL of atreatment as follows:

-   -   1. SLP (50 ml/L); dipentene (50 ml/L); primary amyl acetate (50        ml/L); monoammonium phosphate (10 g/L); sodium citrate (10 g/L);        remainder of volume=Star3+.    -   2. SLP (25 m/L); dipentene (50 ml/L); primary amyl acetate (50        ml/L); monoammonium phosphate (10 g/L); sodium citrate (10 g/L);        remainder of volume=Star3+.    -   3. SLP (50 ml/L); dipentene (50 m/L); primary amyl acetate (50        ml/L); monoammonium phosphate (10 g/L); sodium citrate (10 g/L);        isopropyl alcohol (4 ml/L); remainder of volume=Star3+.    -   4. Same as #3.    -   5. (Positive control) SLP (5 ml/L); ammonium hydroxide (3 mi/L);        isopropyl alcohol (4 ml/L); monoammonium phosphate (2 g/L);        mixture of chelating agents (10 g/L); remainder of volume=water.    -   6. Water.

The amount of oil released from the sands after 22 hours is measured.Due to the non-polar nature of dipentene and primary amyl acetate, whichdo not dissolve in water, the approximate amount of dipentene andprimary amyl acetate present in tubes 1 through 4 (2.5 ml) is subtractedfrom the estimated oil recovery measurement. The results are as follows:

-   -   1. 1.75 ml    -   2. 2 ml    -   3. 1.75 ml    -   4. 1.75 ml    -   5. 3 ml    -   6. 0 ml

Treatment number 5 recovered about 80% of the oil from the sand, withnumber 2 recovering about 55% of the oil.

Example 3—Pour Point Study #1

Crude oil precipitates (i.e., paraffin) extracted from an oil well inTexas, were used to study the capabilities of the subject composition toalter pour point.

To measure the pour point of a sample of the paraffin without treatment,11 g of paraffin was heated gradually in a hot water bath to a maximumtemperature of 87° C. The paraffin was observed for the occurrence ofmelting throughout the gradual heating. Minimal melting occurred at 87°C., so the pour point was determined to be >87° C.

To measure the pour point of the paraffin with treatment, three separatetests were performed, using the ASTM D97 standard pour point testprocedure as a loose guideline. Three samples from the same paraffinwere treated with identical amounts of a composition according to thesubject invention. The samples were then heated preliminarily for 2hours at 35° C.

The samples were mixed and then chilled to allow for formation of theparaffin wax crystals, with examination of the samples for flowcharacteristics occurring at every −3° C. temperature interval. Thelowest temperature at which movement of the sample is observed isrecorded as the pour point.

The mixture in each sample was observed until it became solid, whichoccurred at −3° C. This temperature was determined to be the pour pointfor this particular paraffin when treated with the subject composition.The pour point value was reduced from the pour point measured in theuntreated sample.

As shown in FIG. 1, at −3° C., the mixture of paraffin and treatmentcomposition separated into a lower, solid paraffin portion and a topliquid portion. The liquid portion, comprising D-limonene and canolaoil, separated from the mixture while it was being chilled and remainedin liquid form at the −3° C. temperature.

Example 4—Pour Point Study #2

Paraffinic Texas Permian crude that was treated with the composition ofthe subject invention was tested for pour point depression. The ASTMD5950 standard test method for pour point of petroleum products(automatic tilt method) was used. The resulting pour point value wasabout −25° F., or about −32° C.

Example 5—Paraffin Dispersal

A hard, low-grade paraffin crude was sampled from a well in IrionCountry, Texas and treated with two formulations (1 and 2) of thesubject composition in test tubes. Three replicates (1, 1a, 1b) wereperformed using Formulation 1, and three replicates were performed usingFormulation 2 (2, 2a, 2b).

Formulation 1 comprised: Star 3+ diluted; SLP 10 ml/L; turpentine 50ml/L; monoammonium phosphate 10 g/L; sodium citrate 10 g/L.

Formulation 2 comprised: Star 3+ diluted; SLP 10 ml/L; turpentine 100ml/L; monoammonium phosphate 10 g/L; sodium citrate 10 g/L.

The paraffin grade dispersal grade was for each replicate after 2 hoursand 4 hours, with grade 5 being fully dispersed paraffin. The resultsare reported in Tables 1 and 2, below. See also, FIGS. 2A-2B.

TABLE 1 Increase in paraffin grade over 4 hours when treated withFormulation 1. Grade Time: 1 hr. 2 hr. 3 hr. 4 hr. 1 Formulation 1 4 51a Formulation 1 4 5 1b Formulation 1 4 5

TABLE 2 Increase in paraffin grade over 4 hours when treated withFormulation 2. Grade Time: 1 hr. 2 hr. 3 hr. 4 hr. 2 Formulation 2 4 52a Formulation 2 4 5 2b Formulation 2 4 5

Example 6—Paraffin Dispersal

A very hard paraffin crude (#1) was sampled from a well and treated witha formulation (Formulation 3) of a composition according to anembodiment of the subject invention. The results were compared to otherstandard paraffin dispersants. Multiple replicates were performed foreach treatment.

Formulation 3 comprised: Star3+ in 0.1% oil, diluted; SLP 10 ml/L;dipentene 200 ml/L; sodium citrate 10 g/L; monoammonium phosphate 10g/L; and purified MEL 5 ml/L.

The paraffin dispersal grade was measured for each replicate. All fivereplicates of Formulation 3 treatment produced fully dispersed (grade 5)paraffin in only 1 hour.

The results are reported in Table 3, below. See also, FIGS. 3A-3B.

TABLE 3 Paraffin #1 dispersal grade over 1 hour. Grade Time: 1 hr. 1aFormulation 3 5 1b Formulation 3 5 1c Formulation 3 5 1d Formulation 3 51e Formulation 3 5 1a 100% water 0 1b 100% water 0 1a 100% Xylene 3.5 1b100% Xylene 5 1a 100% Kerosene 3 1b 100% Kerosene 4 1a 100% Pentane 4 1b100% Pentane 5 1a Unknown Dispersant A 2 1b Unknown Dispersant A 2 1cUnknown Dispersant A 2 1a Unknown Dispersant B 1 1b Unknown Dispersant B1 1c Unknown Dispersant B 1 1a Unknown Dispersant C 1 1b UnknownDispersant C 1 1c Unknown Dispersant C 1 1a Condensate 4 1b Condensate 5

A hard paraffin crude (#2, less hard than #1) was sampled from a well,treated with Formulation 3 and compared to other standard paraffintreatments. Multiple replicates were performed for each treatment.

The paraffin dispersal grade was measured for each replicate. All fivereplicates of Formulation 3 treatment produced fully dispersed (grade 5)paraffin by 2 hours. The results are reported in Table 4, below. Seealso, FIGS. 4A-4B.

TABLE 4 Paraffin #2 dispersal grade over 2 hours. Grade Time: 1 hr. 2hr. 2a Formulation 3 4.5 5 2b Formulation 3 5 2c Formulation 3 4 5 2dFormulation 3 4.5 5 2e Formulation 3 3.5 5 2a 100% water 0 0 2b 100%water 0 0 2a 100% Xylene 4 5 2b 100% Xylene 4 5 2a 100% Kerosene 3.5 4.52b 100% Kerosene 3.5 5 2a 100% Pentane 4.5 5 2b 100% Pentane 5 2aUnknown Dispersant A 2 2 2b Unknown Dispersant A 2 2 2c UnknownDispersant A 2 2 2a Unknown Dispersant B 2 2 2b Unknown Dispersant B 2 22c Unknown Dispersant B 2 2 2a Unknown Dispersant C 2 2 2b UnknownDispersant C 2 2 2c Unknown Dispersant C 2 2 2a Condensate 5 2bCondensate 5

A hard paraffin crude (#3, less hard than #1 and #2) was sampled from awell, treated Formulation 3 and compared to other standard paraffintreatments. Multiple replicates were performed for each treatment.

The paraffin dispersal grade was measured for each replicate. All fivereplicates of Formulation 3 treatment produced fully dispersed (grade 5)paraffin by 1 hour. The results are reported in Table 5, below. Seealso, FIGS. 5A-5B.

TABLE 5 Paraffin #3 dispersal grade over 1 hour. Grade Time: 1 hr. 3aFormulation 3 5 3b Formulation 3 5 3c Formulation 3 5 3d Formulation 3 53e Formulation 3 5 3a 100% water 0 3b 100% water 0 3a 100% Xylene 5 3b100% Xylene 5 3a 100% Kerosene 4.5 3b 100% Kerosene 5 3a 100% Pentane 53b 100% Pentane 5 3a Unknown Dispersant A 3 3b Unknown Dispersant A 3 3cUnknown Dispersant A 3 3a Unknown Dispersant B 3.5 3b Unknown DispersantB 3.5 3c Unknown Dispersant B 3.5 3a Unknown Dispersant C 2 3b UnknownDispersant C 2 3c Unknown Dispersant C 2 3a Condensate 5 3b Condensate 5

Example 7—Rust Removal

A piece of corroded casing having rust thereon was sampled from an oilwell in the Appalachian region. The metal was submerged in a compositionaccording to an embodiment of the subject invention without anyagitation for 16 hours. FIG. 6 shows the piece of casing beforetreatment (6A) and after treatment (6B).

Example 8—Bacterial Growth Inhibition

Gram-positive and Gram-negative bacterial strains were cultivated innutrient rich broth media with varying concentrations of a compositionaccording to an embodiment of the subject invention in each respectiveflask (1, 5, and 10% v/v). Initial and final cell concentrations of eachflask were calculated in colony forming unit/milliliter (CFU/ml).

For Gram-negative bacteria, 24 hour cultivation ensured maximal cellconcentration when cultivated at 35° C. Due to a lack of availableshaker space operating at 35° C., the Gram-positive bacteria werecultivated in a shaker at 30° C. Because this was lower than the optimum35° C. temperature for the particular strain of bacteria, 72 hours ofcultivation ensured maximal cell concentration and spore formation atthe lower temperature.

TABLE 6 Bacterial inhibition results, Gram-negative bacteria. 0 hr 24 hrFold Sample CFU/ml CFU/ml Increase B. thailendensis control 3.6 × 10⁶9.0 × 10⁹ 2,500X B. thailendensis + 1% 3.7 × 10⁶ 5.0 × 10⁹ 1,350XComposition B. thailendensis + 5% 2.0 × 10⁶ 4.0 × 10⁸  200X CompositionB. thailendensis + 10% 2.1 × 10⁶ 3.8 × 10⁶    2X Composition

The results for inhibition of the Gram-negative bacteria, Bacillusthailendensis, are shown in Table 6. Significant growth inhibition wasobserved as the composition was added at 1, 5, and 10% v/v. FIG. 7A.

The results for inhibition of the Gram-positive bacteria, Bacillussubtilis subtilis “B1,” are shown in Table 7. Significant growthinhibition was observed when the composition was added at 1, 5, and 10%v/v. FIG. 7B.

TABLE 7 Bacterial inhibition results, Gram-positive bacteria. 0 hr 72 hrFold Sample CFU/ml CFU/ml Increase B1 Control 2.1 × 10⁶ 7.0 × 10⁷  33XB1 + 1% 9.6 × 10⁵ 5.0 × 10⁶ 5.2X Composition B1 + 5% 1.4 × 10⁶ 4.0 × 10⁶ 4X Composition B1 + 10% 1.2 × 10⁶ 2.0 × 10⁴ −60X  Composition

Example 9—Biofilm Formation Inhibition—Direct Contact with Composition

B. subtilis subtilis was cultivated in a flask for 48 hours at 35° C. innutrient rich broth to allow for maximal cell concentrations to bereached. 0.1 ml of the mature culture was spread onto 4 sterile nutrientagar plates.

1, 5, and 10% v/v dilutions of a composition according to an embodimentof the subject invention were prepared using sterilized produced waterfrom an Appalachian oil well as the solvent. 0.02 ml of each dilution(1, 5, and 10%) were added directly to the surface of the bacteria ontheir respective agar plates and were incubated at 35° C. for 24 hours.FIGS. 8A-8C show the 5 treatment plates versus untreated controls.Bacterial growth inhibition was clear at the site of treatmentapplication, with a greater area of inhibition corresponding toincreasing percent volume of composition.

Example 10—Biofilm Disruption—Direct Contact with Composition

B. subtilis subtilis was allowed to cultivate and form a biofilm after24 hours of incubation at 35° C. on a nutrient agar plate. Once thebiofilm was established, 0.05 ml of a composition according to anembodiment of the subject invention was directly added to the surface ofthe biofilm at the center of the agar plate to test for biofilmdegradation. As shown in FIGS. 9A-9B, initial signs of biofilmdegradation were observed within 16 hours.

Example 11—Biofilm Disruption—Indirect Contact with Composition

The effects of the composition on biofilm formation were tested byindirect contact. B. subtilis subtilis was spread onto 4 agar plates.Once the spread was visibly dry, a piece of agar was removed from thecenter of each agar plate and a composition according to an embodimentof the subject invention was added to the “well” that was created. Thetreatment soaked into the surrounding agar.

FIGS. 10A-10C show the treatment plates versus untreated controls. Acircular area of thinning biofilm was observed around the agar wellwhere treatment had soaked into the agar.

We claim:
 1. A composition for use in removing a contaminant from asubterranean formation, an oil and/or gas well, a wellbore and/orequipment associated therewith, the composition comprising one or moresolvents, one or more surfactants, a first yeast fermentation product,one or more chelators, and, optionally, one or more ammonium saltsand/or co-surfactants, wherein the one or more surfactants arebiosurfactants, and the one or more solvents and one or morebiosurfactants are not produced by the yeast of the first yeastfermentation product.
 2. The composition of claim 1, wherein the yeastfermentation product comprises a cultivated yeast selected fromWickerhamomyces anomalus, Starmerella bombicola and Meyerozymaguilliermondii, and/or a growth by-product thereof.
 3. The compositionof claim 1, wherein the one or more solvents are selected from terpenes,terpenoids, non-polar aromatic solvents, acetates, alcohols, and ionicor semi-ionic liquids.
 4. The composition of claim 3, wherein the one ormore solvents include isoamyl acetate, primary amyl acetate, isopropylalcohol, d-limonene, dipentene, and/or turpentine.
 5. The composition ofclaim 3, comprising ammonium hydroxide and/or monoammonium phosphate. 6.The composition of claim 1, wherein the one or more chelators includeEDTA, citric acid and/or sodium citrate.
 7. The composition of claim 1,wherein the one or more biosurfactants include a biosurfactant selectedfrom glycolipids, lipopeptides, fatty acid ester compounds, flavolipids,phospholipids, high-molecular-weight biopolymers, lipoproteins,lipopolysaccharide-protein complexes, and polysaccharide-protein-fattyacid complexes.
 8. The composition of claim 7, comprising one or moreglycolipids selected from rhamnolipids, rhamnose-d-phospholipids,trehalose lipids, trehalose dimycolates, trehalose monomycolates,mannosylerythritol lipids, cellobiose lipids, ustilagic acid, lactonicsophorolipids, and acidic sophorolipids.
 9. The composition of claim 7,comprising one or more lipopeptides selected from surfactin, iturin,fengycin, arthrofactin, viscosin, amphisin, syringomycin, and/orlichenysin.
 10. The composition of claim 7, wherein the one or morebiosurfactants include one or more fatty acid ester compounds having thefollowing chemical formula:

wherein R₁═C₆ to C₂₂ saturated or unsaturated hydrocarbon, or anepoxide, or cyclopropane thereof Y₁═H, C₁-C₅ hydrocarbon, or hydroxyl atany position along R₁ Y₂═H, C₁-C₅ hydrocarbon, or hydroxyl at anyposition along R₁ Y₃═H, C₁-C₅ hydrocarbon, or hydroxyl at any positionalong R₂ Y₄═H, C₁-C₅ hydrocarbon, or hydroxyl at any position along R₂R₂═C₁-C₁₀ saturated or unsaturated, branched or unbranched, hydrocarbon.11. The composition of claim 1, wherein the biosurfactants are inpurified form.
 12. The composition of claim 1, further comprising acarrier, said carrier comprising water, oil and/or brine fluids.
 13. Thecomposition according to claim 1 comprising: water, a first yeastfermentation product comprising cultivated Wickerhamomyces anomalusand/or growth by-products thereof; one or more biosurfactants selectedfrom glycolipids, lipopeptides and esterified fatty acid compounds; oneor more solvents selected from turpentine, dipentene, d-limonene,isoamyl acetate, and/or primary amyl acetate; sodium citrate; and,optionally, ammonium hydroxide and/or monoammonium hydroxide.
 14. Thecomposition of claim 13, comprising: 10 ml/L to 50 ml/L biosurfactants,10 ml/L to 50 ml/L dipentene, 10 ml/L to 50 ml/L isoamyl acetate, 1 g/Lto 10 g/L sodium citrate, and 1 g/L to 10 g/L monoammonium phosphate.15. A method for improving oil and/or gas production wherein acomposition of claim 5 is applied to a subterranean formation, an oiland/or gas well, a wellbore and/or equipment associated therewith,wherein the equipment comprises a pipe line, storage tank, casing,tubing, rod, and/or pump.
 16. The method of claim 15, used to remove acontaminant from the formation, well, wellbore and/or equipment whileenhancing oil recovery, wherein the contaminant is a solid paraffindeposit and/or a paraffin precipitate, wherein the deposit and/orprecipitate comprises paraffin molecules having carbon chain lengths of20 or greater, and wherein the composition dissolves and/or liquefiesthe contaminant.
 17. The method of claim 15, used to disperse and/oremulsify a solid paraffin deposit and/or a paraffin precipitate intocrude oil fluids, wherein the deposit and/or precipitate comprisesparaffin molecules having carbon chain lengths of 20 or greater.
 18. Themethod of claim 15, used to inhibit crystallization of paraffindispersed in crude oil.
 19. The method of claim 15, used to preventdeposition of dispersed paraffin crystals onto surfaces of thesubterranean formation, oil and/or gas well, wellbore and/or equipment.20. The method of claim 15, used to reduce the viscosity of paraffiniccrude oil.
 21. The method of claim 15, used to reduce the pour point ofparaffinic crude oil to about −25° F./−32° C.
 22. The method of claim15, used to remove and/or dissolve scale deposits on surfaces of thesubterranean formation, oil and/or gas well, wellbore and/or equipment.23. The method of claim 15, used to release rust from oilfield casingsand related equipment and to protect against under-deposit rust-relatedcorrosion of the equipment.
 24. The method of claim 15, used to inhibitbacterial growth and disrupt biofilm formation on surfaces of thesubterranean formation, oil and/or gas well, wellbore and/or equipment.25. The method of claim 24, used to protect against microbial inducedcorrosion (MIC).
 26. The method of claim 15, used to remediate formationskin damage, wherein the composition increases the permeability ofnear-wellbore formation by dissolving paraffin matrices in formationpores, and wherein the composition alters the wettability of thenear-wellbore formation so that it is water-wet.
 27. The method of claim15, wherein the well is an offshore well.
 28. The method of claim 15,wherein the method is used in oil wells with formation water salinity of250,000 ppm (total dissolved solids) or more.
 29. The method of claim15, wherein 200 to 1000 gallons of the composition is pumped into andout of a well with shut-in time of 24 hours or less.